Diagnotic markers

ABSTRACT

The present invention provides methods of predicting response to a cancer therapy based on the methylation status of the ERBB2 gene. One aspect of the invention provides a method of predicting response to an EGFR inhibitor therapy based on the methylation status of the ERBB2 gene.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/529,917 filed Aug. 31, 2011, the disclosure of which is incorporatedherein by reference in its entirety.

SEQUENCE LISTING

The present application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 29, 2012, isnamed P4755R1US_ST25.txt and is 5,519 bytes in size.

FIELD OF THE INVENTION

The present invention provides methods of predicting response to acancer therapy based on gene methylation status.

BACKGROUND OF THE INVENTION

The present invention is directed to methods for diagnosing and treatingcancer patients. In particular, the present invention is directed tomethods for determining which patients will most benefit from treatmentwith an epidermal growth factor receptor (EGFR) kinase inhibitor.

Cancer is a generic name for a wide range of cellular malignanciescharacterized by unregulated growth, lack of differentiation, and theability to invade local tissues and metastasize. These neoplasticmalignancies affect, with various degrees of prevalence, every tissueand organ in the body.

A multitude of therapeutic agents have been developed over the past fewdecades for the treatment of various types of cancer. The most commonlyused types of anticancer agents include: DNA-alkylating agents (e.g.,cyclophosphamide, ifosfamide), antimetabolites (e.g., methotrexate, afolate antagonist, and 5-fluorouracil, a pyrimidine antagonist),microtubule disrupters (e.g., vincristine, vinblastine, paclitaxel), DNAintercalators (e.g., doxorubicin, daunomycin, cisplatin), and hormonetherapy (e.g., tamoxifen, flutamide).

The epidermal growth factor receptor (EGFR) family comprises fourclosely related receptors (HER1/EGFR, HER2 (ERBB2), HER3 (ERBB23), andHER4 (ERBB4)) involved in cellular responses such as differentiation andproliferation. Over-expression of the EGFR kinase, or its ligandTGF-alpha, is frequently associated with many cancers, including breast,lung, colorectal, ovarian, renal cell, bladder, head and neck cancers,glioblastomas, and astrocytomas, and is believed to contribute to themalignant growth of these tumors. A specific deletion-mutation in theEGFR gene (EGFRvIII) has also been found to increase cellulartumorigenicity. Activation of EGFR stimulated signaling pathways promotemultiple processes that are potentially cancer-promoting, e.g.proliferation, angiogenesis, cell motility and invasion, decreasedapoptosis and induction of drug resistance. Increased HER1/EGFRexpression is frequently linked to advanced disease, metastases and poorprognosis. For example, in NSCLC and gastric cancer, increased HER1/EGFRexpression has been shown to correlate with a high metastatic rate, poortumor differentiation and increased tumor proliferation.

ERBB2 overexpression is commonly regarded as a predictor of a poorprognosis, especially in patients with primary disease that involvesaxillary lymph nodes ((Slamon et al., Science 235:177-182 (1987); Slamonet al., Science 244:707-712 (1989); Ravdin and Chamness, Gene 159:19-27(1995); and Hynes and Stem, Biochim Biophys Acta 1198:165-184 (1994),and has been linked to sensitivity and/or resistance to hormone therapyand chemotherapeutic regimens, including CMF (cyclophosphamide,methotrexate, and fluoruracil) and anthracyclines (Baselga et al.,Oncology 11(3 Suppl 2):43-48 (1997)). Patients treated with the HER2antibody trastuzumab are selected for therapy based on HER2overexpression/amplification. See, for example, WO99/31140,US2003/0170234, WO01/89566.

Mutations which activate the receptor's intrinsic protein tyrosinekinase activity and/or increase downstream signaling have been observedin NSCLC and glioblastoma. However the role of mutations as a principlemechanism in conferring sensitivity to EGF receptor inhibitors, forexample erlotinib (TARCEVA®) or gefitinib (IRESSA™), has beencontroversial. Recently, a mutant form of the full length EGF receptorhas been reported to predict responsiveness to the EGF receptor tyrosinekinase inhibitor gefitinib (Paez, J. G. et al. (2004) Science304:1497-1500; Lynch, T. J. et al. (2004) N. Engl. J. Med.350:2129-2139). Cell culture studies have shown that cell lines whichexpress the mutant form of the EGF receptor (i.e. H3255) were moresensitive to growth inhibition by the EGF receptor tyrosine kinaseinhibitor gefitinib, and that much higher concentrations of gefitinibwas required to inhibit the tumor cell lines expressing wild type EGFreceptor. These observations suggests that specific mutant forms of theEGF receptor may reflect a greater sensitivity to EGF receptorinhibitors but do not identify a completely non-responsive phenotype.

The development for use as anti-tumor agents of compounds that directlyinhibit the kinase activity of the EGFR, as well as antibodies thatreduce EGFR kinase activity by blocking EGFR activation, are areas ofintense research effort (de Bono J. S. and Rowinsky, E. K. (2002) Trendsin Mol. Medicine. 8:S19-S26; Dancey, J. and Sausville, E. A. (2003)Nature Rev. Drug Discovery 2:92-313). Several studies have demonstrated,disclosed, or suggested that some EGFR kinase inhibitors might improvetumor cell or neoplasia killing when used in combination with certainother anti-cancer or chemotherapeutic agents or treatments (e.g. Herbst,R. S. et al. (2001) Expert Opin. Biol. Ther. 1:719-732; Solomon, B. etal (2003) Int. J. Radiat. Oncol. Biol. Phys. 55:713-723; Krishnan, S. etal. (2003) Frontiers in Bioscience 8, e1-13; Grunwald, V. and Hidalgo,M. (2003) J. Nat. Cancer Inst. 95:851-867; Seymour L. (2003) CurrentOpin. Investig. Drugs 4(6):658-666; Khalil, M. Y. et al. (2003) ExpertRev. Anticancer Ther. 3:367-380; Bulgaru, A. M. et al. (2003) ExpertRev. Anticancer Ther. 3:269-279; Dancey, J. and Sausville, E. A. (2003)Nature Rev. Drug Discovery 2:92-313; Ciardiello, F. et al. (2000) Clin.Cancer Res. 6:2053-2063; and Patent Publication No: US 2003/0157104).

Erlotinib (e.g. erlotinib HCl, also known as TARCEVA® or OSI-774) is anorally available inhibitor of EGFR kinase. In vitro, erlotinib hasdemonstrated substantial inhibitory activity against EGFR kinase in anumber of human tumor cell lines, including colorectal and breast cancer(Moyer J. D. et al. (1997) Cancer Res. 57:4838), and preclinicalevaluation has demonstrated activity against a number of EGFR-expressinghuman tumor xenografts (Pollack, V. A. et al (1999) J. Pharmacol. Exp.Ther. 291:739). More recently, erlotinib has demonstrated promisingactivity in phase I and II trials in a number of indications, includinghead and neck cancer (Soulieres, D., et al. (2004) J. Clin. Oncol.22:77), NSCLC (Perez-Soler R, et al. (2001) Proc. Am. Soc. Clin. Oncol.20:310a, abstract 1235), CRC (Oza, M., et al. (2003) Proc. Am. Soc.Clin. Oncol. 22:196a, abstract 785) and MBC (Winer, E., et al. (2002)Breast Cancer Res. Treat. 76:5115a, abstract 445). In a phase III trial,erlotinib monotherapy significantly prolonged survival, delayed diseaseprogression and delayed worsening of lung cancer-related symptoms inpatients with advanced, treatment-refractory NSCLC (Shepherd, F. et al.(2004) J. Clin. Oncology, 22:14 S (July 15 Supplement), Abstract 7022).While much of the clinical trial data for erlotinib relate to its use inNSCLC, preliminary results from phase I/II studies have demonstratedpromising activity for erlotinib and capecitabine/erlotinib combinationtherapy in patients with wide range of human solid tumor types,including CRC (Oza, M., et al. (2003) Proc. Am. Soc. Clin. Oncol.22:196a, abstract 785) and MBC (Jones, R. J., et al. (2003) Proc. Am.Soc. Clin. Oncol. 22:45a, abstract 180). In November 2004 the U.S. Foodand Drug Administration (FDA) approved erlotinib for the treatment ofpatients with locally advanced or metastatic non-small cell lung cancer(NSCLC) after failure of at least one prior chemotherapy regimen.Erlotinib is the only drug in the epidermal growth factor receptor(EGFR) class to demonstrate in a Phase III clinical trial an increase insurvival in advanced NSCLC patients.

An anti-neoplastic drug would ideally kill cancer cells selectively,with a wide therapeutic index relative to its toxicity towardsnon-malignant cells. It would also retain its efficacy against malignantcells, even after prolonged exposure to the drug. Unfortunately, none ofthe current chemotherapies possess such an ideal profile. Instead, mostpossess very narrow therapeutic indexes. Furthermore, cancerous cellsexposed to slightly sub-lethal concentrations of a chemotherapeuticagent will very often develop resistance to such an agent, and quiteoften cross-resistance to several other antineoplastic agents as well.Additionally, for any given cancer type one frequently cannot predictwhich patient is likely to respond to a particular treatment, even withnewer gene-targeted therapies, such as EGFR kinase inhibitors, thusnecessitating considerable trial and error, often at considerable riskand discomfort to the patient, in order to find the most effectivetherapy.

Thus, there is a need for more efficacious treatment for neoplasia andother proliferative disorders, and for more effective means fordetermining which tumors will respond to which treatment. Strategies forenhancing the therapeutic efficacy of existing drugs have involvedchanges in the schedule for their administration, and also their use incombination with other anticancer or biochemical modulating agents.Combination therapy is well known as a method that can result in greaterefficacy and diminished side effects relative to the use of thetherapeutically relevant dose of each agent alone. In some cases, theefficacy of the drug combination is additive (the efficacy of thecombination is approximately equal to the sum of the effects of eachdrug alone), but in other cases the effect is synergistic (the efficacyof the combination is greater than the sum of the effects of each druggiven alone).

Target-specific therapeutic approaches, such as erlotinib, are generallyassociated with reduced toxicity compared with conventional cytotoxicagents, and therefore lend themselves to use in combination regimens.Promising results have been observed in phase I/II studies of erlotinibin combination with bevacizumab (Mininberg, E. D., et al. (2003) Proc.Am. Soc. Clin. Oncol. 22:627a, abstract 2521) and gemcitabine(Dragovich, T., (2003) Proc. Am. Soc. Clin. Oncol. 22:223a, abstract895). Recent data in NSCLC phase III trials have shown that first-lineerlotinib or gefitinib in combination with standard chemotherapy did notimprove survival (Gatzemeier, U., (2004) Proc. Am. Soc. Clin. Oncol.23:617 (Abstract 7010); Herbst, R. S., (2004) Proc. Am. Soc. Clin.Oncol. 23:617 (Abstract 7011); Giaccone, G., et al. (2004) J. Clin.Oncol. 22:777; Herbst, R., et al. (2004) J. Clin. Oncol. 22:785).However, pancreatic cancer phase III trials have shown that first-lineerlotinib in combination with gemcitabine did improve survival.

Several groups have investigated potential biomarkers to predict apatient's response to EGFR inhibitors (see for example, WO 2004/063709,WO 2005/017493, WO 2004/111273, WO 2004/071572; US 2005/0019785, and US2004/0132097). One such biomarker is epithelial and mesenchymalphenotype. During most cancer metastases, an important change occurs ina tumor cell known as the epithelial-to-mesenchymal transition (EMT)(Thiery, J. P. (2002) Nat. Rev. Cancer 2:442-454; Savagner, P. (2001)Bioessays 23:912-923; Kang Y. and Massague, J. (2004) Cell 118:277-279;Julien-Grille, S., et al. Cancer Research 63:2172-2178; Bates, R. C. etal. (2003) Current Biology 13:1721-1727; Lu Z., et al. (2003) CancerCell. 4(6):499-515)). Epithelial cells, which are bound together tightlyand exhibit polarity, give rise to mesenchymal cells, which are heldtogether more loosely, exhibit a loss of polarity, and have the abilityto travel. These mesenchymal cells can spread into tissues surroundingthe original tumor, invade blood and lymph vessels, and travel to newlocations where they divide and form additional tumors. EMT does notoccur in healthy cells except during embryogenesis. Under normalcircumstances TGF-β acts as a growth inhibitor, however, during cancermetastasis, TGF-β begins to promote EMT.

Epithelial and mesenchymal phenotypes have been associated withparticular gene expression patterns. For example, epithelial phenotypewas shown in WO2006101925 to be associated with high expression levelsof E-cadherin, Brk, γ-catenin, α-catenin, keratin 8, keratin 18,connexin 31, plakophilin 3, stratafin 1, laminin alpha-5 and ST14whereas mesenchymal phenotype was associated with high expression levelsof vimentin, fibronectin, fibrillin-1, fibrillin-2, collagenalpha-2(IV), collagen alpha-2(V), LOXL1, nidogen, C11orf9, tenascin,N-cadherin, embryonal EDB+ fibronectin, tubulin alpha-3 and epimorphin.

Epigenetics is the study of heritable changes in gene expression orcellular phenotype caused by mechanisms other than changes in theunderlying DNA sequence—hence the name epi-(Greek: over, above,outer)-genetics. Examples of such changes include DNA methylation andhistone modifications, both of which serve to modulate gene expressionwithout altering the sequence of the associated genes. These changes canbe somatically heritable through cell division for the remainder of thelife of the organism and may also be passed on to subsequent generationsof the organism. However, there is no change in the underlying DNAsequence of the organism; instead, non-genetic factors cause theorganism's genes to behave or express differently.

DNA methylation is a crucial part of normal organismal development andcellular differentiation in higher organisms. DNA methylation stablyalters the gene expression pattern in cells such that cells can“remember where they have been”; for example, cells programmed to bepancreatic islets during embryonic development remain pancreatic isletsthroughout the life of the organism without continuing signals tellingthem that they need to remain islets. In addition, DNA methylationsuppresses the expression of viral genes and other deleterious elementsthat have been incorporated into the genome of the host over time. DNAmethylation also forms the basis of chromatin structure, which enablescells to form the myriad characteristics necessary for multicellularlife from a single immutable sequence of DNA. DNA methylation also playsa crucial role in the development of nearly all types of cancer. DNAmethylation at the 5 position of cytosine has the specific effect ofreducing gene expression and has been found in every vertebrateexamined. In adult somatic tissues, DNA methylation typically occurs ina CpG dinucleotide context while non-CpG methylation is prevalent inembryonic stem cells.

“CpG” is shorthand for “-C-phosphate-G-”, that is, cytosine and guanineseparated by only one phosphate; phosphate links any two nucleosidestogether in DNA. The “CpG” notation is used to distinguish this linearsequence from the CG base-pairing of cytosine and guanine. Cytosines inCpG dinucleotides can be methylated to form 5-methylcytosine (5-mC). Inmammals, methylating the cytosine within a gene can turn the gene offEnzymes that add a methyl group to DNA are called DNAmethyltransferases. In mammals, 70% to 80% of CpG cytosines aremethylated. There are regions of the genome that have a higherconcentration of CpG sites, known as CpG islands. These “CpG islands”also have a higher than expected GC content (i.e. >50%). Many genes inmammalian genomes have CpG islands associated with the start of thegene. Because of this, the presence of a CpG island is used to help inthe prediction and annotation of genes. CpG islands are refractory tomethylation, which may help maintain an open chromatin configuration. Inaddition, this could result in a reduced vulnerability to transitionmutations and, as a consequence, a higher equilibrium density of CpGssurviving. Methylation of CpG sites within the promoters of genes canlead to their silencing, a feature found in a number of human cancers(for example the silencing of tumor suppressor genes). In contrast, thehypomethylation of CpG sites has been associated with theover-expression of oncogenes within cancer cells.

SUMMARY OF THE INVENTION

One aspect of the invention provides for a method of determining thesensitivity of tumor cell growth to inhibition by an EGFR kinaseinhibitor, comprising detecting the methylation status of the ERBB2 genein a sample tumor cell, wherein hypomethylation of the ERBB2 geneindicates that the tumor cell growth is sensitive to inhibition with theEGFR inhibitor. Another aspect of the invention provides for a method ofidentifying a cancer patient who is likely to benefit from treatmentwith an EFGR inhibitor comprising detecting the methylation status ofthe ERBB2 gene from a sample from the patient's cancer, wherein thepatient is identified as being likely to benefit from treatment with theEGFR inhibitor if the methylation status of the ERBB2 gene is detectedto be hypomethylation. In one embodiment, the patient is administered atherapeutically effective amount of an EGFR inhibitor if the patient isidentified as one who will likely benefit from treatment with the EGFRinhibitor.

Another aspect of the invention provides for a method of treating acancer in a patient comprising administering a therapeutically effectiveamount of an EGFR inhibitor to the patient, wherein the patient, priorto administration of the EGFR inhibitor, was diagnosed with a cancerwhich exhibits hypomethylation of the ERBB2 gene, wherein thehypomethylation of the ERBB2 gene is indicative of therapeuticresponsiveness by the subject to the EGFR inhibitor.

Another aspect of the invention provides for a method of selecting atherapy for a cancer patient, comprising the steps of detecting themethylation status of the ERBB2 gene from a sample from the patient'scancer, and selecting an EGFR inhibitor for the therapy when the ERBB2gene is detected to be hypomethylated. In one embodiment, the patient isadministered a therapeutically effective amount of the EGFR inhibitor,such as, for example, erlotinib, cetuximab, or panitumumab.

Another aspect of the invention provides for a method of determiningoverexpression of ERBB2 gene in a cell comprising detecting themethylation status of the ERBB2 gene in the cell, wherein ERBB2 genehypomethylation indicates overexpression of ERBB2 in the cell.

Another aspect of the invention provides for a method treating a cancerin a patient comprising administering a therapeutically effective amountof a HER2 inhibitor to the patient, wherein the patient, prior toadministration of the HER2 inhibitor, was diagnosed with a cancer whichexhibits hypomethylation of the ERBB2 gene, wherein the hypomethylationof the ERBB2 gene is indicative of therapeutic responsiveness by thesubject to the HER2 inhibitor.

Another aspect of the invention provides for a method of selecting atherapy for a cancer patient, comprising the steps of detecting themethylation status of the ERBB2 gene from a sample from the patient'scancer, and selecting a HER2 inhibitor for the therapy when the ERBB2gene is detected to be hypomethylated. In one embodiment, the patient isadministered a therapeutically effective amount of the HER2 inhibitor,such as trastuzumab or T-DM1.

In certain embodiments of the above methods, the methylation status isdetected in a part of the ERBB2 gene. The part of the gene used todetect the methylation status is, for example, an enhancer region, or anenhancer region and a promoter region. In one embodiment, the part ofthe gene used to detect the methylation status comprises the nucleicacid sequence of SEQ ID NO:1. In one embodiment, the part of the geneused to detect the methylation status consists of the nucleic acidsequence of SEQ ID NO:1. In one embodiment, the part of the gene used todetect the methylation status comprises a 6 CpG repeat region. In oneembodiment, the part of the gene used to detect the methylation statuscomprises the nucleic acid sequence of SEQ ID NO: 2. In one embodiment,the part of the gene used to detect the methylation status consists ofthe nucleic acid sequence of SEQ ID NO: 2.

In certain embodiments of the methods, the methylation status of theERBB2 gene is deemed to a hypomethylation status if the ERBB2 gene, orpart thereof, is less than about 50% or less than about 20% methylated.

In certain embodiments of the above methods, the methylation status isdetected by pyrosequencing. In certain embodiments of the above methods,the ERBB2 gene is from a formalin-fixed paraffin embedded (FFPE) tissueor from fresh frozen tissue. In certain embodiments of the abovemethods, the ERBB2 gene isolated from the tissue sample is preamplifiedbefore pyrosequencing.

In certain embodiments of the above methods, the tumor cell is a NSCLCtumor cell or the cancer is NSCLC.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the nucleic acid sequence of the ERBB2 enhancer region (SEQID NO: 1) containing 28 CpG methylation sites (SEQ ID NO: 1).

FIG. 2 is a graph depicting the results of a pyrosequencing analysis ofERBB2 methylation status in NSCLC surgically resected primary tumors andmatched normal tissue.

FIG. 3 is a graph depicting the results of a quantitative pyrosequencinganalysis of ERBB2 methylation status in epithelial-like andmesenchymal-like NSCLC cell lines.

FIG. 4 is a graph showing the relative expression of ERBB2 mRNA in NSCLCcells using TaqMan based Fludigm gene expression analysis.

FIG. 5 is a graph depicting percent methylation of ERBB2 enhancer CpGsites in cells lines. The cells lines are ordered by sensitivity toerlotinib treatment.

FIG. 6 is a graph depicting the results of a pyrosequencing analysisshowing percentages of methylation at each of 6 individual CpG sites inNSCLC surgically resected primary tumors and matched normal tissue.

FIG. 7 is a graph depicting percent methylation of ERBB2 enhancer regionin high and low ERBB2-expression tumor cells.

FIG. 8 is a graph depicting pyrosequencing analysis of methylation ofERBB2 and epithelial/mesenchymal status in 47 NSCLC primary tumorsamples derived from archival FFPE slides.

DESCRIPTION OF THE INVENTION I. Definitions

Unless otherwise defined, all terms of art, notations and otherscientific terminology used herein are intended to have the meaningscommonly understood by those of skill in the art to which this inventionpertains. In some cases, terms with commonly understood meanings aredefined herein for clarity and/or for ready reference, and the inclusionof such definitions herein should not necessarily be construed torepresent a substantial difference over what is generally understood inthe art. The techniques and procedures described or referenced hereinare generally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized molecular cloning methodologies described in Sambrook etal., Molecular Cloning: A Laboratory Manual 2nd. edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. As appropriate,procedures involving the use of commercially available kits and reagentsare generally carried out in accordance with manufacturer definedprotocols and/or parameters unless otherwise noted.

Before the present methods, kits and uses therefore are described, it isto be understood that this invention is not limited to the particularmethodology, protocols, cell lines, animal species or genera,constructs, and reagents described as such may, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise.

Throughout this specification and claims, the word “comprise,” orvariations such as “comprises” or “comprising,” will be understood toimply the inclusion of a stated integer or group of integers but not theexclusion of any other integer or group of integers.

The term “cancer” in an animal refers to the presence of cellspossessing characteristics typical of cancer-causing cells, such asuncontrolled proliferation, immortality, metastatic potential, rapidgrowth and proliferation rate, and certain characteristic morphologicalfeatures. Often, cancer cells will be in the form of a tumor, but suchcells may exist alone within an animal, or may circulate in the bloodstream as independent cells, such as leukemic cells.

“Abnormal cell growth”, as used herein, unless otherwise indicated,refers to cell growth that is independent of normal regulatorymechanisms (e.g., loss of contact inhibition). This includes theabnormal growth of: (1) tumor cells (tumors) that proliferate byexpressing a mutated tyrosine kinase or overexpression of a receptortyrosine kinase; (2) benign and malignant cells of other proliferativediseases in which aberrant tyrosine kinase activation occurs; (4) anytumors that proliferate by receptor tyrosine kinases; (5) any tumorsthat proliferate by aberrant serine/threonine kinase activation; and (6)benign and malignant cells of other proliferative diseases in whichaberrant serine/threonine kinase activation occurs.

The term “treating” as used herein, unless otherwise indicated, meansreversing, alleviating, inhibiting the progress of, or preventing,either partially or completely, the growth of tumors, tumor metastases,or other cancer-causing or neoplastic cells in a patient.

The term “treatment” as used herein, unless otherwise indicated, refersto the act of treating.

The phrase “a method of treating” or its equivalent, when applied to,for example, cancer refers to a procedure or course of action that isdesigned to reduce or eliminate the number of cancer cells in an animal,or to alleviate the symptoms of a cancer.

“A method of treating” cancer or another proliferative disorder does notnecessarily mean that the cancer cells or other disorder will, in fact,be eliminated, that the number of cells or disorder will, in fact, bereduced, or that the symptoms of a cancer or other disorder will, infact, be alleviated.

The term “therapeutically effective agent” means a composition that willelicit the biological or medical response of a tissue, system, animal orhuman that is being sought by the researcher, veterinarian, medicaldoctor or other clinician.

The term “therapeutically effective amount” or “effective amount” meansthe amount of the subject compound or combination that will elicit thebiological or medical response of a tissue, system, animal or human thatis being sought by the researcher, veterinarian, medical doctor or otherclinician.

The terms “ErbB1”, “HER1”, “epidermal growth factor receptor” and “EGFR”and “EGFR kinase” are used interchangeably herein and refer to EGFR asdisclosed, for example, in Carpenter et al. Ann. Rev. Biochem.56:881-914 (1987), including naturally occurring mutant forms thereof(e.g. a deletion mutant EGFR as in Humphrey et al. PNAS (USA)87:4207-4211 (1990)). erbB1 refers to the gene encoding the EGFR proteinproduct.

As used herein, the term “EGFR kinase inhibitor” and “EGFR inhibitor”refers to any EGFR kinase inhibitor that is currently known in the artor that will be identified in the future, and includes any chemicalentity that, upon administration to a patient, results in inhibition ofa biological activity associated with activation of the EGF receptor inthe patient, including any of the downstream biological effectsotherwise resulting from the binding to EGFR of its natural ligand. SuchEGFR kinase inhibitors include any agent that can block EGFR activationor any of the downstream biological effects of EGFR activation that arerelevant to treating cancer in a patient. Such an inhibitor can act bybinding directly to the intracellular domain of the receptor andinhibiting its kinase activity. Alternatively, such an inhibitor can actby occupying the ligand binding site or a portion thereof of the EGFreceptor, thereby making the receptor inaccessible to its natural ligandso that its normal biological activity is prevented or reduced.Alternatively, such an inhibitor can act by modulating the dimerizationof EGFR polypeptides, or interaction of EGFR polypeptide with otherproteins, or enhance ubiquitination and endocytotic degradation of EGFR.EGFR kinase inhibitors include but are not limited to low molecularweight inhibitors, antibodies or antibody fragments, antisenseconstructs, small inhibitory RNAs (i.e. RNA interference by dsRNA;RNAi), and ribozymes. In a preferred embodiment, the EGFR kinaseinhibitor is a small organic molecule or an antibody that bindsspecifically to the human EGFR.

Inhibitors of EGF receptor function have shown clinical utility and thedefinition of key EGF receptor signaling pathways which describe patientsubsets most likely to benefit from therapy has become an important areaof investigation. Mutations which activate the receptor's intrinsicprotein tyrosine kinase activity and/or increase downstream signalinghave been observed in NSCLC and glioblastoma. In vitro and clinicalstudies have shown considerable variability between wt EGF receptor celllines and tumors in their cellular responses to EGF receptor inhibition,which in part has been shown to derive from EGF receptor independentactivation of the phosphatidyl inositol 3-kinase pathway, leading to thecontinued phosphorylation of the anti-apoptotic serine-threonine kinaseAkt. The molecular determinants to alternative routes of PI3-kinaseactivation and consequent EGF receptor inhibitor insensitivity are anactive area of investigation. For example the insulin-like growthfactor-1 receptor (IGF-1 receptor), which strongly activates thePI3-kinase pathway, has been implicated in cellular resistance to EGFinhibitors. The roles of cell-cell and cell-adhesion networks, which canalso exert survival signals through the PI3-kinase pathway in mediatinginsensitivity to selective EGF receptor inhibition are less clear andwould be postulated to impact cell sensitivity to EGF receptor blockade.The ability of tumor cells to maintain growth and survival signals inthe absence of adhesion to extracellular matrix or cell-cell contacts isimportant not only in the context of cell migration and metastasis butalso in maintaining cell proliferation and survival in wound-like tumorenvironments where extracellular matrix is being remodeled and cellcontact inhibition is diminished. We previously defined an EMT geneexpression signature that correlates with in vitro sensitivity of NSCLCcell lines to erlotinib (Yauch et al., 2005, Clin Cancer Res 11,8686-8698).

The expressions “ErbB2” and “HER2” are used interchangeably herein andrefer to human HER2 protein described, for example, in Semba et al.,PNAS (USA) 82:6497-6501 (1985) and Yamamoto et al. Nature 319:230-234(1986) (Genebank accession number X03363). The term “erbB2” refers tothe gene encoding human ErbB2 and “neu” refers to the gene encoding ratp185neu.

“ErbB3” and “HER3” refer to the receptor polypeptide as disclosed, forexample, in U.S. Pat. Nos. 5,183,884 and 5,480,968 as well as Kraus etal. PNAS (USA) 86:9193-9197 (1989).

The terms “ErbB4” and “HER4” herein refer to the receptor polypeptide asdisclosed, for example, in EP Pat Appin No 599,274; Plowman et al.,Proc. Natl. Acad. Sci. USA, 90:1746-1750 (1993); and Plowman et al.,Nature, 366:473-475 (1993), including isoforms thereof, e.g., asdisclosed in WO99/19488, published Apr. 22, 1999.

By “hypomethylation” is meant that a majority of the possibly methylatedCpG sites are unmethylated. In certain embodiments, hypomethylationmeans that less than 50%, less than 45%, less than 40%, less than 35%,less than 30%, less than 25%, less than 20%, less than 15%, less than10%, less than 5%, or less than 1% of the possible methylation sites ina part of the ERBB2 gene is methylated. In one embodiment, the part ofthe EBB2 gene comprises an enhancer region of ERBB2. In one embodiment,the part of the EBB2 gene comprises the ERBB2 enhancer region of SEQ IDNO: 1, containing 28 CpG methylation sites. In yet another embodiment,hypomethylation means that fewer possible methylation sites aremethylated compared to an ERBB2 gene that is expressed at a normallevel, for example, in a non-tumor cell. In another embodiment,hypomethylation means that none of the CpG sites in the enhancer regionof the ERBB2 gene is methylated.

II. Methods and Compositions

The present invention relates, in part, to the discovery thathypomethylation of the ERBB2 gene correlates with high expression ofERBB2 and sensitivity of cancers to treatment with EGFR kinaseinhibitors. Accordingly, the present invention provides a method ofdetermining the sensitivity of a tumor to cell growth inhibition by anEGFR kinase inhibitor in a cancer patient, comprising obtaining a sampleof the tumor and analyzing the tumor sample to detect methylation statusof ERBB2, wherein detection of a hypomethylation status of ERBB2indicates that the tumor cell growth is sensitive to inhibition by EGFRinhibitor treatment.

Accordingly, in one embodiment, there is provided a method ofdetermining sensitivity of tumor cell growth to inhibition by an EGFRkinase inhibitor comprising detecting the methylation status of theERBB2 gene in a sample tumor cell, wherein hypomethylation of the ERBB2gene indicates that the tumor cell growth is sensitive to inhibitionwith the EGFR inhibitor.

Another aspect of the invention provides for a method of identifying acancer patient who is likely to benefit from treatment with an EFGRinhibitor comprising detecting the methylation status of the ERBB2 genefrom a sample from the patient's cancer, such as a sample from acancerous tumor, wherein the patient is identified as being likely tobenefit from treatment with the EGFR inhibitor if the ERBB2 gene isdetected to be hypomethylated. In some embodiments, the patient isadministered a therapeutically effective amount of the EGFR inhibitorbased on the hypomethylation status.

Furthermore, provided herein are methods of identifying an patient whois more likely to exhibit benefit from a therapy with an EGFR inhibitor,the method comprising detecting hypomethylation in a part of the ERBB2gene, wherein less than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%,10%, 5%, 2%, or 1% methylation of the analyzed part of the ERBB2 genesequence indicates that the patient is more likely to benefit fromtreatment.

Another aspect of the invention provides for a method of selecting atherapy for a cancer patient based on the methylation status of theERBB2 gene in a sample taken from the patent's cancer, such as a sampleof a cancerous tumor. In one embodiment, the method of selecting atherapy comprises the steps of detecting the methylation status of theERBB2 gene from a sample from the patient's cancer, and selecting anEGFR inhibitor for the therapy when the ERBB2 gene is detected to behypomethylated (the methylation status is deemed to be a hypomethylatedstatus). The patient is then administered a therapeutically effectiveamount of the EGFR inhibitor based on this selection method. In someembodiments, the EFGR inhibitor is erlotinib, cetuximab, or panitumumab.

Another aspect of the invention provides for a method of treating apatient with an EGFR inhibitor if the patient is suffering from a cancercharacterized by ERBB2 hypomethylation.

Yet another aspect of the invention provides for a method of determiningwhether the ERBB2 gene is overexpress in a cell, such as a cancer cell,comprising detecting the methylation status of the ERBB2 gene in thecell, wherein a determination that the ERBB2 gene is hypomethylatedindicates overexpression of ERBB2 in the cell.

Overexpression of the ERBB2 gene has been previously correlated toresponse to HER2 inhibitors, such as trastuzumab (HERCEPTIN®, Genentech,Inc.) and T-DM1. See, for example, WO99/31140, US2003/0170234,WO01/89566. As such, another aspect of the invention provides for amethod of treating a patient with an HER2 inhibitor if the patient issuffering from a cancer characterized by ERBB2 hypomethylation.Accordingly, provided herein are methods of identifying an individualwho is more likely to exhibit benefit from a therapy comprising an HER2inhibitor, the method comprising detecting hypomethylation in a part ofthe ERBB2 gene, wherein less than about 50%, 45%, 40%, 35%, 30%, 25%,20%, 15%, 10%, 5%, 2%, or 1% methylation of the ERBB2 sequence indicatesthat the individual is more likely to benefit from treatment.

In one embodiment, the invention provides a method of treating cancer ina patient comprising administering a therapeutically effective amount ofa HER2 inhibitor to the patient, wherein the patient, prior toadministration of the HER2 inhibitor, was diagnosed with a cancercharacterized by ERBB2 hypomethylation, wherein the ERBB2hypomethylation is indicative of therapeutic responsiveness by thesubject to the HER2 inhibitor. In one embodiment, the HER2 inhibitor isa small molecule or an antibody. In one embodiment, the HER2 inhibitoris an antibody such as trastuzumab or T-DM1.

Another aspect of the invention provides for a method of selecting atherapy for a cancer patient, comprising the steps of detecting themethylation status of the ERBB2 gene from a sample from the patient'scancer and selecting a HER2 inhibitor for the therapy when the ERBB2gene is detected to be hypomethylated.

In some embodiments of the above methods of detecting methylation statusof the ERBB2 gene, the part of the ERBB2 gene analyzed for methylationstatus comprises an enhancer region. In some embodiments, the part ofthe ERBB2 gene comprises an enhancer region and a promoter region. Insome embodiments, the part of the ERBB2 gene is part of the gene at orcomprising the chromosomal location of chr17:37,861,100-37,863,650 (NCBIbuild 37/hg19). In some embodiments, the part of the ERBB2 gene is thesequence represented by SEQ ID NO:1. In some embodiments, the part ofthe ERBB2 gene comprises a 6 CpG repeat region of SEQ ID NO:1. In oneembodiment, the part of the ERBB2 gene comprises the 6 CpG repeat regionof SEQ ID NO: 2.

In some embodiments, the part of the ERBB2 gene is pre-amplified priorto quantitative methylation specific PCR.

In certain embodiments of the above methods, the methylation status ofthe ERBB2 gene or a specific part of the gene is deemed to behypomethylated when less than about 50%, 45%, 40%, 35%, 30%, 25%, 20%,15%, 10%, 5%, 2%, or 1% methylation of the analyzed part of the ERBB2gene sequence is detected.

Presence and/or level/amount of various biomarkers in a sample can beanalyzed by a number of methodologies, many of which are known in theart and understood by the skilled artisan, including, but not limitedto, immunohistochemical (“IHC”), Western blot analysis,immunoprecipitation, molecular binding assays, ELISA, ELIFA,fluorescence activated cell sorting (“FACS”), MassARRAY, proteomics,quantitative blood based assays (as for example Serum ELISA),biochemical enzymatic activity assays, in situ hybridization, Southernanalysis, Northern analysis, whole genome sequencing, polymerase chainreaction (“PCR”) including quantitative real time PCR (“qRT-PCR”) andother amplification type detection methods, such as, for example,branched DNA, SISBA, TMA and the like), RNA-Seq, FISH, microarrayanalysis, gene expression profiling, and/or serial analysis of geneexpression (“SAGE”), as well as any one of the wide variety of assaysthat can be performed by protein, gene, and/or tissue array analysis.Typical protocols for evaluating the status of genes and gene productsare found, for example in Ausubel et al., eds., 1995, Current ProtocolsIn Molecular Biology, Units 2 (Northern Blotting), 4 (SouthernBlotting), 15 (Immunoblotting) and 18 (PCR Analysis). Multiplexedimmunoassays such as those available from Rules Based Medicine or MesoScale Discovery (“MSD”) may also be used.

Methods for evaluation of DNA methylation are well known. For example,Laird (2010) Nature Reviews Genetics 11:191-203 provides a review of DNAmethylation analysis. In some embodiments, methods for evaluatingmethylation include randomly shearing or randomly fragmenting thegenomic DNA, cutting the DNA with a methylation-dependent ormethylation-sensitive restriction enzyme and subsequently selectivelyidentifying and/or analyzing the cut or uncut DNA. Selectiveidentification can include, for example, separating cut and uncut DNA(e.g., by size) and quantifying a sequence of interest that was cut or,alternatively, that was not cut. See, e.g., U.S. Pat. No. 7,186,512. Insome embodiments, the method can encompass amplifying intact DNA afterrestriction enzyme digestion, thereby only amplifying DNA that was notcleaved by the restriction enzyme in the area amplified. See, e.g., U.S.patent application Ser. Nos. 10/971,986; 11/071,013; and 10/971,339. Insome embodiments, amplification can be performed using primers that aregene specific. Alternatively, adaptors can be added to the ends of therandomly fragmented DNA, the DNA can be digested with amethylation-dependent or methylation-sensitive restriction enzyme,intact DNA can be amplified using primers that hybridize to the adaptorsequences. In some embodiments, a second step can be performed todetermine the presence, absence or quantity of a particular gene in anamplified pool of DNA. In some embodiments, the DNA is amplified usingreal-time, quantitative PCR.

In some embodiments, the methods comprise quantifying the averagemethylation density in a target sequence within a population of genomicDNA. In some embodiments, the method comprises contacting genomic DNAwith a methylation-dependent restriction enzyme or methylation-sensitiverestriction enzyme under conditions that allow for at least some copiesof potential restriction enzyme cleavage sites in the locus to remainuncleaved; quantifying intact copies of the locus; and comparing thequantity of amplified product to a control value representing thequantity of methylation of control DNA, thereby quantifying the averagemethylation density in the locus compared to the methylation density ofthe control DNA.

The quantity of methylation of a locus of DNA can be determined byproviding a sample of genomic DNA comprising the locus, cleaving the DNAwith a restriction enzyme that is either methylation-sensitive ormethylation-dependent, and then quantifying the amount of intact DNA orquantifying the amount of cut DNA at the DNA locus of interest. Theamount of intact or cut DNA will depend on the initial amount of genomicDNA containing the locus, the amount of methylation in the locus, andthe number (i.e., the fraction) of nucleotides in the locus that aremethylated in the genomic DNA. The amount of methylation in a DNA locuscan be determined by comparing the quantity of intact DNA or cut DNA toa control value representing the quantity of intact DNA or cut DNA in asimilarly-treated DNA sample. The control value can represent a known orpredicted number of methylated nucleotides. Alternatively, the controlvalue can represent the quantity of intact or cut DNA from the samelocus in another (e.g., normal, non-diseased) cell or a second locus.

By using methylation-sensitive or methylation-dependent restrictionenzyme under conditions that allow for at least some copies of potentialrestriction enzyme cleavage sites in the locus to remain uncleaved andsubsequently quantifying the remaining intact copies and comparing thequantity to a control, average methylation density of a locus can bedetermined. If the methylation-sensitive restriction enzyme is contactedto copies of a DNA locus under conditions that allow for at least somecopies of potential restriction enzyme cleavage sites in the locus toremain uncleaved, then the remaining intact DNA will be directlyproportional to the methylation density, and thus may be compared to acontrol to determine the relative methylation density of the locus inthe sample. Similarly, if a methylation-dependent restriction enzyme iscontacted to copies of a DNA locus under conditions that allow for atleast some copies of potential restriction enzyme cleavage sites in thelocus to remain uncleaved, then the remaining intact DNA will beinversely proportional to the methylation density, and thus may becompared to a control to determine the relative methylation density ofthe locus in the sample. Such assays are disclosed in, e.g., U.S. patentapplication Ser. No. 10/971,986.

In some embodiments, quantitative amplification methods (e.g.,quantitative PCR or quantitative linear amplification) can be used toquantify the amount of intact DNA within a locus flanked byamplification primers following restriction digestion. Methods ofquantitative amplification are disclosed in, e.g., U.S. Pat. Nos.6,180,349; 6,033,854; and 5,972,602, as well as in, e.g., Gibson et al.,Genome Research 6:995-1001 (1996); DeGraves et al., Biotechniques34(1):106-10, 112-5 (2003); Deiman B et al., Mol. Biotechnol.20(2):163-79 (2002).

Additional methods for detecting DNA methylation can involve genomicsequencing before and after treatment of the DNA with bisulfite. See,e.g., Frommer et al., Proc. Natl. Acad. Sci. USA 89:1827-1831 (1992).When sodium bisulfite is contacted to DNA, unmethylated cytosine isconverted to uracil, while methylated cytosine is not modified.

In some embodiments, restriction enzyme digestion of PCR productsamplified from bisulfite-converted DNA is used to detect DNAmethylation. See, e.g., Sadri & Hornsby, Nucl. Acids Res. 24:5058-5059(1996); Xiong & Laird, Nucleic Acids Res. 25:2532-2534 (1997).

In some embodiments, a MethyLight assay is used alone or in combinationwith other methods to detect DNA methylation (see, Eads et al., CancerRes. 59:2302-2306 (1999)). Briefly, in the MethyLight process genomicDNA is converted in a sodium bisulfite reaction (the bisulfite processconverts unmethylated cytosine residues to uracil). Amplification of aDNA sequence of interest is then performed using PCR primers thathybridize to CpG dinucleotides. By using primers that hybridize only tosequences resulting from bisulfite conversion of unmethylated DNA, (oralternatively to methylated sequences that are not converted)amplification can indicate methylation status of sequences where theprimers hybridize. Similarly, the amplification product can be detectedwith a probe that specifically binds to a sequence resulting frombisulfite treatment of an unmethylated (or methylated) DNA. If desired,both primers and probes can be used to detect methylation status. Thus,kits for use with MethyLight can include sodium bisulfite as well asprimers or detectably-labeled probes (including but not limited toTaqman or molecular beacon probes) that distinguish between methylatedand unmethylated DNA that have been treated with bisulfite. Other kitcomponents can include, e.g., reagents necessary for amplification ofDNA including but not limited to, PCR buffers, deoxynucleotides; and athermostable polymerase.

In some embodiments, a Ms-SNuPE (Methylation-sensitive Single NucleotidePrimer Extension) reaction is used alone or in combination with othermethods to detect DNA methylation (see Gonzalgo & Jones Nucleic AcidsRes. 25:2529-2531 (1997)). The Ms-SNuPE technique is a quantitativemethod for assessing methylation differences at specific CpG sites basedon bisulfite treatment of DNA, followed by single-nucleotide primerextension. Briefly, genomic DNA is reacted with sodium bisulfite toconvert unmethylated cytosine to uracil while leaving 5-methylcytosineunchanged. Amplification of the desired target sequence is thenperformed using PCR primers specific for bisulfite-converted DNA, andthe resulting product is isolated and used as a template for methylationanalysis at the CpG site(s) of interest.

In some embodiments, a methylation-specific PCR (“MSP”) reaction is usedalone or in combination with other methods to detect DNA methylation. AnMSP assay entails initial modification of DNA by sodium bisulfite,converting all unmethylated, but not methylated, cytosines to uracil,and subsequent amplification with primers specific for methylated versusunmethylated DNA. See, Herman et al., Proc. Natl. Acad. Sci. USA93:9821-9826, (1996); U.S. Pat. No. 5,786,146. In some embodiments, DNAmethylation is detected by a QIAGEN PyroMark CpG Assay predesignedPyrosequencing DNA Methylation assays.

In some embodiments, cell methylation status is determined usinghigh-throughput DNA methylation analysis to determine sensitivity toEGFR inhibitors. Briefly, genomic DNA is isolated from a cell or tissuesample (e.g. a tumor sample or a blood sample) and is converted in asodium bisulfite reaction (the bisulfite process converts unmethylatedcytosine residues to uracil) using standard assays in the art. Thebisulfite converted DNA product is amplified, fragmented and hybridizedto an array containing CpG sites from across a genome using standardassays in the art. Following hybridization, the array is imaged andprocessed for analysis of the DNA methylation status using standardassays in the art. In some embodiments, the tissue sample isformalin-fixed paraffin embedded (FFPE) tissue. In some embodiments, thetissue sample is fresh frozen tissue. In some embodiments, the DNAisolated from the tissue sample is preamplified before bisulfiteconversion. In some embodiments, the DNA isolated from the tissue sampleis preamplified before bisulfite conversion by using the InvitrogenSuperscript III One-Step RT-PCR System with Platinum Taq. In someembodiments, the DNA isolated from the tissue sample is preamplifiedbefore bisulfite conversion using a Taqman based assay. In someembodiments, the sodium bisulfite reaction is conducted using the ZymoEZ DNA Methylation Kit. In some embodiments, the bisulfite converted DNAis amplified and hybridized to an array using the Illumina InfiniumHumanMethylation450 Beadchip Kit. In some embodiments, the array isimaged on an Illumina iScan Reader. In some embodiments, the images areprocessed with the GenomeStudio software methylation module. In someembodiments, the methylation data is analyzed using the Bioconductorlumi software package. See Du et al., Bioinformatics, 24(13):1547-1548(2008).

In some embodiments, ERBB2 DNA methylation sites are identified usingbisulfite sequencing PCR (BSP) to determine sensitivity to EGFRinhibitors. Briefly, genomic DNA is isolated from a cell or tissuesample (e.g., a tumor sample or a blood sample) and is converted in asodium bisulfite reaction (the bisulfite process converts unmethylatedcytosine residues to uracil) using standard assays in the art. Thebisulfite converted DNA product is amplified using primers designed tobe specific to the bisulfite converted DNA (e.g., bisulfite-specificprimers) and ligated into vectors for transformation into a host cellusing standard assays in the art. After selection of the host cellscontaining the PCR amplified bisulfite converted DNA product ofinterest, the DNA product is isolated and sequenced to determine thesites of methylation using standard assays in the art. In someembodiments, the tissue sample is formalin-fixed paraffin embedded(FFPE) tissue. In some embodiments, the tissue sample is an FFPE tissuethat has been processed for IHC analysis; for example, for ERBB2expression. In some embodiments, the tissue sample is an FFPE tissuethat showed little or no ERBB2 expression by IHC. In some embodiments,the tissue sample is fresh frozen tissue. In some embodiments, the DNAisolated from the tissue sample is preamplified before bisulfiteconversion. In some embodiments, the DNA isolated from the tissue sampleis preamplified before bisulfite conversion using the InvitrogenSuperscript III One-Step RT-PCR System with Platinum Taq. In someembodiments, the DNA isolated from the tissue sample is preamplifiedbefore bisulfite conversion using a Taqman based assay. In someembodiments, the sodium bisulfite reaction is conducted using the ZymoEZ DNA Methylation-Gold Kit. In some embodiments, the primers designedto be specific to the bisulfite converted DNA are designed using AppliedBiosystems Methyl Primer Express software. In some embodiments, thebisulfite converted DNA product is PCR amplified using the InvitrogenSuperscript III One-Step RT-PCR System with Platinum Taq. In furtherembodiments, the PCR amplified bisulfite converted DNA product isligated into a vector using the Invitrogen TOPO TA Cloning kit. In someembodiments, the host cell is bacteria. In some embodiments, theisolated PCR amplified bisulfite converted DNA product of interest issequenced using Applied Biosystems 3730×1 DNA Analyzer. In someembodiments, the primers designed to be specific to the bisulfiteconverted DNA are designed using Qiagen PyroMark Assay Design software.In some embodiments, the bisulfite converted DNA product is PCRamplified using the Invitrogen Superscript III One-Step RT-PCR Systemwith Platinum Taq. In further embodiments, the PCR amplified bisulfiteconverted DNA product is sequenced using Qiagen Pyromark Q24 andanalyzed Qiagen with PyroMark software.

In some embodiments, ERBB2 DNA methylation sites are identified usingquantitative methylation specific PCR (QMSP) to determine sensitivity toEGFR or HER2 inhibitors. Briefly, genomic DNA is isolated from a cell ortissue sample and is converted in a sodium bisulfite reaction (thebisulfite process converts unmethylated cytosine residues to uracil)using standard assays in the art. In some embodiments, the tissue sampleis formalin-fixed paraffin embedded (FFPE) tissue. In some embodiments,the tissue sample is an FFPE tissue that has been processed for IHCanalysis; for example, for ERBB2 expression. In some embodiments, thetissue sample is an FFPE tissue that showed little or no ERBB2expression by IHC. In some embodiments, the tissue sample is freshfrozen tissue. The bisulfite converted DNA product is amplified usingprimers designed to be specific to the bisulfite converted DNA (e.g.,quantitative methylation specific PCR primers). The bisulfite convertedDNA product is amplified with quantitative methylation specific PCRprimers and analyzed for methylation using standard assays in the art.In some embodiments, the tissue sample is formalin-fixed paraffinembedded (FFPE) tissue. In some embodiments, the tissue sample is freshfrozen tissue. In some embodiments, the DNA isolated from the tissuesample is preamplified before bisulfite conversion using the InvitrogenSuperscript III One-Step RT-PCR System with Platinum Taq. In someembodiments, the DNA isolated from the tissue sample is preamplifiedbefore bisulfite conversion. In some embodiments, the DNA isolated fromthe tissue sample is preamplified before bisulfite conversion using aTaqman based assay. In some embodiments, the sodium bisulfite reactionis conducted using a commercially available kit. In some embodiments,the sodium bisulfite reaction is conducted using the Zymo EZ DNAMethylation-Gold Kit. In some embodiments, the primers designed to bespecific to the bisulfite converted DNA are designed using AppliedBiosystems Methyl Primer Express software. In some embodiments, thebisulfite converted DNA is amplified using a Taqman based assay. In someembodiments, the bisulfite converted DNA is amplified on an AppliedBiosystems 7900HT and analyzed using Applied Biosystems SDS software.

In some embodiments, the invention provides methods to determine ERBB2methylation by 1) IHC analysis of tumor samples, followed by 2)quantitative methylation specific PCR of DNA extracted from the tumortissue used in the IHC analysis of step 1. Briefly, coverslips from IHCslides are removed by one of two methods: the slide are placed in afreezer for at least 15 minutes, then the coverslip is pried off of themicroscope slide using a razor blade. Slides are then incubated inxylene at room temp to dissolve the mounting media. Alternatively,slides are soaked in xylene until the coverslip falls off. This can takeup to several days. All slides are taken through a deparaffinizationprocedure of 5 min xylene (x3), and 5 min 100% ethanol (x2). Tissues arescraped off slides with razor blades and placed in a tissue lysis buffercontaining proteinase K and incubated overnight at 56° C. In cases wheretissue is still present after incubation, an extra 10 μl Proteinase Kmay be added and the tissue is incubated for another 30 min. DNAextraction was continued; for example, by using a QIAamp DNA FFPE Tissuekit. DNA extracted directly from IHC slides was subject to QMSP analysisusing the QMSP3 primers and probes as described above.

In some embodiments, the bisulfite-converted DNA is sequenced by a deepsequencing. Deep sequencing is a process, such as direct pyrosequencing,where a sequence is read multiple times. Deep sequencing can be used todetect rare events such as rare mutations. Ultra-deep sequencing of alimited number of loci may been achieved by direct pyrosequencing of PCRproducts and by sequencing of more than 100 PCR products in a singlerun. A challenge in sequencing bisulphite-converted DNA arises from itslow sequence complexity following bisulfite conversion of cytosineresidues to thymine (uracil) residues. Reduced representation bisulphitesequencing (RRBS) may be introduced to reduce sequence redundancy byselecting only some regions of the genome for sequencing bysize-fractionation of DNA fragments (Laird, P W Nature Reviews11:195-203 (2010)). Targeting may be accomplished by array capture orpadlock capture before sequencing. For example, targeted capture onfixed arrays or by solution hybrid selection can enrich for sequencestargeted by a library of DNA or RNA oligonucleotides and can beperformed before or after bisulphite conversion. Alternatively, padlockcapture provides improved enrichment efficiency by combining theincreased annealing specificity of two tethered probes, and subsequentamplification with universal primers allows for a more uniformrepresentation than amplification with locus-specific primers.

Additional methylation detection methods include, but are not limitedto, methylated CpG island amplification (see Toyota et al., Cancer Res.59:2307-12 (1999)) and those described in, e.g., U.S. Patent Publication2005/0069879; Rein et al., Nucleic Acids Res. 26 (10): 2255-64 (1998);Olek et al., Nat Genet. 17(3): 275-6 (1997); Laird, P W Nature Reviews11:195-203 (2010); and PCT Publication No. WO 00/70090).

In some embodiments, the expression of ERBB2 in a cell is determined byevaluating ERBB2 mRNA in a cell. Methods for the evaluation of mRNAs incells are well known and include, for example, hybridization assaysusing complementary DNA probes (such as in situ hybridization usinglabeled riboprobes specific for the one or more genes, Northern blot andrelated techniques) and various nucleic acid amplification assays (suchas RT-PCR using complementary primers specific for one or more of thegenes, and other amplification type detection methods, such as, forexample, branched DNA, SISBA, TMA and the like). In some embodiments,the expression of ERBB2 in a test sample is compared to a referencesample. For example, the test sample may be a tumor tissue sample andthe reference sample may be from normal tissue or cells such as PBMCs.

Samples from mammals can be conveniently assayed for mRNAs usingNorthern, dot blot or PCR analysis. In addition, such methods caninclude one or more steps that allow one to determine the levels oftarget mRNA in a biological sample (e.g., by simultaneously examiningthe levels a comparative control mRNA sequence of a “housekeeping” genesuch as an actin family member). Optionally, the sequence of theamplified target cDNA can be determined.

Optional methods of the invention include protocols which examine ordetect mRNAs, such as target mRNAs, in a tissue or cell sample bymicroarray technologies. Using nucleic acid microarrays, test andcontrol mRNA samples from test and control tissue samples are reversetranscribed and labeled to generate cDNA probes. The probes are thenhybridized to an array of nucleic acids immobilized on a solid support.The array is configured such that the sequence and position of eachmember of the array is known. For example, a selection of genes whoseexpression correlates with increased or reduced clinical benefit ofanti-angiogenic therapy may be arrayed on a solid support. Hybridizationof a labeled probe with a particular array member indicates that thesample from which the probe was derived expresses that gene.

According to some embodiments, presence and/or level/amount is measuredby observing protein expression levels of an aforementioned gene. Incertain embodiments, the method comprises contacting the biologicalsample with antibodies to a biomarker described herein under conditionspermissive for binding of the biomarker, and detecting whether a complexis formed between the antibodies and biomarker. Such method may be an invitro or in vivo method.

In certain embodiments, the presence and/or level/amount of biomarkerproteins in a sample are examined using IHC and staining protocols. IHCstaining of tissue sections has been shown to be a reliable method ofdetermining or detecting presence of proteins in a sample. In oneaspect, level of biomarker is determined using a method comprising: (a)performing IHC analysis of a sample (such as a subject cancer sample)with an antibody; and b) determining level of a biomarker in the sample.In some embodiments, IHC staining intensity is determined relative to areference value.

IHC may be performed in combination with additional techniques such asmorphological staining and/or fluorescence in-situ hybridization. Twogeneral methods of IHC are available; direct and indirect assays.According to the first assay, binding of antibody to the target antigenis determined directly. This direct assay uses a labeled reagent, suchas a fluorescent tag or an enzyme-labeled primary antibody, which can bevisualized without further antibody interaction. In a typical indirectassay, unconjugated primary antibody binds to the antigen and then alabeled secondary antibody binds to the primary antibody. Where thesecondary antibody is conjugated to an enzymatic label, a chromogenic orfluorogenic substrate is added to provide visualization of the antigen.Signal amplification occurs because several secondary antibodies mayreact with different epitopes on the primary antibody.

The primary and/or secondary antibody used for IHC typically will belabeled with a detectable moiety. Numerous labels are available whichcan be generally grouped into the following categories: (a)Radioisotopes, such as ³⁵S, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I; (b) colloidal goldparticles; (c) fluorescent labels including, but are not limited to,rare earth chelates (europium chelates), Texas Red, rhodamine,fluorescein, dansyl, Lissamine, umbelliferone, phycocrytherin,phycocyanin, or commercially available fluorophores such SPECTRUMORANGE7 and SPECTRUM GREEN7 and/or derivatives of any one or more of theabove; (d) various enzyme-substrate labels are available and U.S. Pat.No. 4,275,149 provides a review of some of these. Examples of enzymaticlabels include luciferases (e.g., firefly luciferase and bacterialluciferase; U.S. Pat. No. 4,737,456), luciferin,2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidasesuch as horseradish peroxidase (HRPO), alkaline phosphatase,β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g.,glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocyclic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase, and the like.

Examples of enzyme-substrate combinations include, for example,horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate;alkaline phosphatase (AP) with para-Nitrophenyl phosphate as chromogenicsubstrate; and β-D-galactosidase (β-D-Gal) with a chromogenic substrate(e.g., p-nitrophenyl-β-D-galactosidase) or fluorogenic substrate (e.g.,4-methylumbelliferyl-β-D-galactosidase). For a general review of these,see U.S. Pat. Nos. 4,275,149 and 4,318,980.

Specimens thus prepared may be mounted and coverslipped. Slideevaluation is then determined, e.g., using a microscope, and stainingintensity criteria, routinely used in the art, may be employed. In someembodiments, a staining pattern score of about 1+ or higher isdiagnostic and/or prognostic. In certain embodiments, a staining patternscore of about 2+ or higher in an IHC assay is diagnostic and/orprognostic. In other embodiments, a staining pattern score of about 3 orhigher is diagnostic and/or prognostic. In one embodiment, it isunderstood that when cells and/or tissue from a tumor or colon adenomaare examined using IHC, staining is generally determined or assessed intumor cell and/or tissue (as opposed to stromal or surrounding tissuethat may be present in the sample).

In alternative methods, the sample may be contacted with an antibodyspecific for the biomarker under conditions sufficient for anantibody-biomarker complex to form, and then detecting the complex. Thepresence of the biomarker may be detected in a number of ways, such asby Western blotting and ELISA procedures for assaying a wide variety oftissues and samples, including plasma or serum. A wide range ofimmunoassay techniques using such an assay format are available, see,e.g., U.S. Pat. Nos. 4,016,043, 4,424,279 and 4,018,653. These includeboth single-site and two-site or “sandwich” assays of thenon-competitive types, as well as in the traditional competitive bindingassays. These assays also include direct binding of a labeled antibodyto a target biomarker.

Presence and/or level/amount of a selected biomarker in a tissue or cellsample may also be examined by way of functional or activity-basedassays. For instance, if the biomarker is an enzyme, one may conductassays known in the art to determine or detect the presence of the givenenzymatic activity in the tissue or cell sample.

In certain embodiments, the samples are normalized for both differencesin the amount of the biomarker assayed and variability in the quality ofthe samples used, and variability between assay runs. Such normalizationmay be accomplished by detecting and incorporating the level of certainnormalizing biomarkers, including well known housekeeping genes, such asACTB. Alternatively, normalization can be based on the mean or mediansignal of all of the assayed genes or a large subset thereof (globalnormalization approach). On a gene-by-gene basis, measured normalizedamount of a subject tumor mRNA or protein is compared to the amountfound in a reference set. Normalized expression levels for each mRNA orprotein per tested tumor per subject can be expressed as a percentage ofthe expression level measured in the reference set. The presence and/orexpression level/amount measured in a particular subject sample to beanalyzed will fall at some percentile within this range, which can bedetermined by methods well known in the art.

In certain embodiments, relative expression level of a gene isdetermined as follows:

Relative expression gene1 sample1=2exp (Ct housekeeping gene−Ct gene1)with Ct determined in a sample.

Relative expression gene1 reference RNA=2exp (Ct housekeeping gene−Ctgene1) with Ct determined in the reference sample.

Normalized relative expression gene1 sample1=(relative expression gene1sample1/relative expression gene1 reference RNA)×100

Ct is the threshold cycle. The Ct is the cycle number at which thefluorescence generated within a reaction crosses the threshold line.

All experiments are normalized to a reference RNA, which is acomprehensive mix of RNA from various tissue sources (e.g., referenceRNA #636538 from Clontech, Mountain View, Calif.). Identical referenceRNA is included in each qRT-PCR run, allowing comparison of resultsbetween different experimental runs.

In one embodiment, the sample is a clinical sample. In anotherembodiment, the sample is used in a diagnostic assay. In someembodiments, the sample is obtained from a primary or metastatic tumor.Tissue biopsy is often used to obtain a representative piece of tumortissue. Alternatively, tumor cells can be obtained indirectly in theform of tissues or fluids that are known or thought to contain the tumorcells of interest. For instance, samples of lung cancer lesions may beobtained by resection, bronchoscopy, fine needle aspiration, bronchialbrushings, or from sputum, pleural fluid or blood. In some embodiments,the sample includes circulating tumor cells; for example, circulatingcancer cells in blood, urine or sputum. Genes or gene products can bedetected from cancer or tumor tissue or from other body samples such asurine, sputum, serum or plasma. The same techniques discussed above fordetection of target genes or gene products in cancerous samples can beapplied to other body samples. Cancer cells may be sloughed off fromcancer lesions and appear in such body samples. By screening such bodysamples, a simple early diagnosis can be achieved for these cancers. Inaddition, the progress of therapy can be monitored more easily bytesting such body samples for target genes or gene products.

In certain embodiments, a reference sample, reference cell, referencetissue, control sample, control cell, or control tissue is a singlesample or combined multiple samples from the same subject or individualthat are obtained at one or more different time points than when thetest sample is obtained. For example, a reference sample, referencecell, reference tissue, control sample, control cell, or control tissueis obtained at an earlier time point from the same subject or individualthan when the test sample is obtained. Such reference sample, referencecell, reference tissue, control sample, control cell, or control tissuemay be useful if the reference sample is obtained during initialdiagnosis of cancer and the test sample is later obtained when thecancer becomes metastatic.

In certain embodiments, a reference sample, reference cell, referencetissue, control sample, control cell, or control tissue is a combinedmultiple samples from one or more healthy individuals who are not thesubject or individual. In certain embodiments, a reference sample,reference cell, reference tissue, control sample, control cell, orcontrol tissue is a combined multiple samples from one or moreindividuals with a disease or disorder (e.g., cancer) who are not thesubject or individual. In certain embodiments, a reference sample,reference cell, reference tissue, control sample, control cell, orcontrol tissue is pooled RNA samples from normal tissues or pooledplasma or serum samples from one or more individuals who are not thesubject or individual. In certain embodiments, a reference sample,reference cell, reference tissue, control sample, control cell, orcontrol tissue is pooled RNA samples from tumor tissues or pooled plasmaor serum samples from one or more individuals with a disease or disorder(e.g., cancer) who are not the subject or individual.

In the methods of this invention, the tissue samples may be bodilyfluids or excretions such as blood, urine, saliva, stool, pleural fluid,lymphatic fluid, sputum, ascites, prostatic fluid, cerebrospinal fluid(CSF), or any other bodily secretion or derivative thereof. By blood itis meant to include whole blood, plasma, serum or any derivative ofblood. Assessment of tumor epithelial or mesenchymal biomarkers in suchbodily fluids or excretions can sometimes be preferred in circumstanceswhere an invasive sampling method is inappropriate or inconvenient.

In the methods of this invention, the tumor cell can be a lung cancertumor cell (e.g. non-small cell lung cancer (NSCLC)), a pancreaticcancer tumor cell, a breast cancer tumor cell, a head and neck cancertumor cell, a gastric cancer tumor cell, a colon cancer tumor cell, anovarian cancer tumor cell, or a tumor cell from any of a variety ofother cancers as described herein below. The tumor cell is preferably ofa type known to or expected to express EGFR, as do all tumor cells fromsolid tumors. The EGFR kinase can be wild type or a mutant form.

In the methods of this invention, the tumor can be a lung cancer tumor(e.g. non-small cell lung cancer (NSCLC)), a pancreatic cancer tumor, abreast cancer tumor, a head and neck cancer tumor, a gastric cancertumor, a colon cancer tumor, an ovarian cancer tumor, or a tumor fromany of a variety of other cancers as described herein below. The tumoris preferably of a type whose cells are known to or expected to expressEGFR, as do all solid tumors. The EGFR can be wild type or a mutantform.

Inhibitors and Pharmaceutical Compositions

Exemplary EGFR kinase inhibitors suitable for use in the inventioninclude, for example quinazoline EGFR kinase inhibitors,pyrido-pyrimidine EGFR kinase inhibitors, pyrimido-pyrimidine EGFRkinase inhibitors, pyrrolo-pyrimidine EGFR kinase inhibitors,pyrazolo-pyrimidine EGFR kinase inhibitors, phenylamino-pyrimidine EGFRkinase inhibitors, oxindole EGFR kinase inhibitors, indolocarbazole EGFRkinase inhibitors, phthalazine EGFR kinase inhibitors, isoflavone EGFRkinase inhibitors, quinalone EGFR kinase inhibitors, and tyrphostin EGFRkinase inhibitors, such as those described in the following patentpublications, and all pharmaceutically acceptable salts and solvates ofthe EGFR kinase inhibitors: International Patent Publication Nos. WO96/33980, WO 96/30347, WO 97/30034, WO 97/30044, WO 97/38994, WO97/49688, WO 98/02434, WO 97/38983, WO 95/19774, WO 95/19970, WO97/13771, WO 98/02437, WO 98/02438, WO 97/32881, WO 98/33798, WO97/32880, WO 97/3288, WO 97/02266, WO 97/27199, WO 98/07726, WO97/34895, WO 96/31510, WO 98/14449, WO 98/14450, WO 98/14451, WO95/09847, WO 97/19065, WO 98/17662, WO 99/35146, WO 99/35132, WO99/07701, and WO 92/20642; European Patent Application Nos. EP 520722,EP 566226, EP 787772, EP 837063, and EP 682027; U.S. Pat. Nos.5,747,498, 5,789,427, 5,650,415, and 5,656,643; and German PatentApplication No. DE 19629652. Additional non-limiting examples of lowmolecular weight EGFR kinase inhibitors include any of the EGFR kinaseinhibitors described in Traxler, P., 1998, Exp. Opin. Ther. Patents8(12):1599-1625.

Specific preferred examples of low molecular weight EGFR kinaseinhibitors that can be used according to the present invention include[6,7-bis(2-methoxyethoxy)-4-quinazolin-4-yl]-(3-ethynylphenyl)amine(also known as OSI-774, erlotinib, or TARCEVA™ (erlotinib HCl); OSIPharmaceuticals/Genentech/Roche) (U.S. Pat. No. 5,747,498; InternationalPatent Publication No. WO 01/34574, and Moyer, J. D. et al. (1997)Cancer Res. 57:4838-4848); CI-1033 (formerly known as PD183805; Pfizer)(Sherwood et al., 1999, Proc. Am. Assoc. Cancer Res. 40:723); PD-158780(Pfizer); AG-1478 (University of California); CGP-59326 (Novartis);PKI-166 (Novartis); EKB-569 (Wyeth); GW-2016 (also known as GW-572016 orlapatinib ditosylate; GSK); and gefitinib (also known as ZD1839 orIRESSA™; Astrazeneca) (Woodburn et al., 1997, Proc. Am. Assoc. CancerRes. 38:633). A particularly preferred low molecular weight EGFR kinaseinhibitor that can be used according to the present invention is[6,7-bis(2-methoxyethoxy)-4-quinazolin-4-yl]-(3-ethynylphenyl)amine(i.e. erlotinib), its hydrochloride salt (i.e. erlotinib HCl, TARCEVA™),or other salt forms (e.g. erlotinib mesylate).

Antibody-based EGFR kinase inhibitors include any anti-EGFR antibody orantibody fragment that can partially or completely block EGFR activationby its natural ligand. Non-limiting examples of antibody-based EGFRkinase inhibitors include those described in Modjtahedi, H., et al.,1993, Br. J. Cancer 67:247-253; Teramoto, T., et al., 1996, Cancer77:639-645; Goldstein et al., 1995, Clin. Cancer Res. 1:1311-1318;Huang, S. M., et al., 1999, Cancer Res. 15:59(8):1935-40; and Yang, X.,et al., 1999, Cancer Res. 59:1236-1243. Thus, the EGFR kinase inhibitorcan be the monoclonal antibody Mab E7.6.3 (Yang, X. D. et al. (1999)Cancer Res. 59:1236-43), or Mab C225 (ATCC Accession No. HB-8508), or anantibody or antibody fragment having the binding specificity thereof.Suitable monoclonal antibody EGFR kinase inhibitors include, but are notlimited to, IMC-C225 (also known as cetuximab or ERBITUX™; ImcloneSystems), ABX-EGF (Abgenix), EMD 72000 (Merck KgaA, Darmstadt), RH3(York Medical Bioscience Inc.), and MDX-447 (Medarex/Merck KgaA).

A variety of HER2 inhibitors are known in the art. These inhibitorsinclude anti-HER2 antibodies. Such antibodies are preferably monoclonalantibodies. They may either be so-called chimaeric antibodies, humanizedantibodies or fully human antibodies. Examples of humanized anti-HER2antibodies are known under the INN names Trastuzumab and Pertuzumab.Trastuzumab is sold by Genentech Inc. and F. Hoffmann-La Roche Ltd underthe trade name HERCEPTIN®. Trastuzumab is an antibody that has antigenbinding residues of, or derived from, the murine 4D5 antibody (ATCC CRL10463, deposited with American Type Culture Collection, 12301 ParklawnDrive, Rockville, Md. 20852 under the Budapest Treaty on May 24, 1990).Exemplary humanized 4D5 antibodies include huMAb4D5-1, huMAb4D5-2,huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 andhuMAb4D5-8 (HERCEPTIN®) as in U.S. Pat. No. 5,821,337.

Another suitable anti-HER2 antibody is trastuzumab-MCC-DM1 (T-DM1), anantibody-drug conjugate (CAS Reg. No. 139504-50-0), which has thestructure:

where Tr is trastuzumab, linked through linker moiety MCC, to themaytansinoid drug moiety, DM1 (U.S. Pat. No. 5,208,020; U.S. Pat. No.6,441,163). The drug to antibody ratio or drug loading is represented byp in the above structure of trastuzumab-MCC-DM1, and ranges in integervalues from 1 to about 8. The drug loading value p is 1 to 8.Trastuzumab-MCC-DM1 includes all mixtures of variously loaded andattached antibody-drug conjugates where 1, 2, 3, 4, 5, 6, 7, and 8 drugmoieties are covalently attached to the antibody trastuzumab (U.S. Pat.No. 7,097,840; US 2005/0276812; US 2005/0166993).

Other HER2 antibodies with various properties have been described inTagliabue et al., Int. J. Cancer, 47:933-937 (1991); McKenzie et al.,Oncogene, 4:543-548 (1989); Cancer Res., 51:5361-5369 (1991); Bacus etal., Molecular Carcinogenesis, 3:350-362 (1990); Stancovski et al., PNAS(USA), 88:8691-8695 (1991); Bacus et al, Cancer Research, 52:2580-2589(1992); Xu et al., Int. J. Cancer, 53:401-408 (1993); WO94/00136;Kasprzyk et al., Cancer Research, 52:2771-2776 (1992); Hancock et al.,Cancer Res., 51:4575-4580 (1991); Shawver et al., Cancer Res.,54:1367-1373 (1994); Arteaga et al., Cancer Res., 54:3758-3765 (1994);Harwerth et al., J. Biol. Chem., 267:15160-15167 (1992); U.S. Pat. No.5,783,186; and Klapper et al., Oncogene, 14:2099-2109 (1997). Furtherdetails on the HER2 antigen and antibodies directed thereto aredescribed in many patent and non-patent publications (for a suitableoverview see U.S. Pat. No. 5,821,337 and WO 2006/044908).

The methods of this invention can be extended to those compounds whichinhibit EGFR and an additional target. These compounds are referred toherein as “bispecific inhibitors”. In one embodiment, the bispecificinhibitor is a bispecific HER3/EGFR, EGFR/HER2, EGFR/HER4 or EGFR c-Met,inhibitor. In one embodiment, the bispecific inhibitor is a bispecificantibody. In one embodiment, the bispecific inhibitor is a bispecificantibody which comprises an antigen binding domain that specificallybinds to EGFR and a second target. In one embodiment, the bispecificinhibitor is a bispecific antibody which comprises an antigen bindingdomain that specifically binds to HER3 and EGFR. In one embodiment, thebispecific HER3/EGFR inhibitor is a bispecific antibody which comprisestwo identical antigen binding domains. Such antibodies are described inU.S. Pat. No. 8,193,321, 20080069820, WO2010108127, US20100255010 andSchaefer et al, Cancer Cell, 20: 472-486 (2011). In one embodiment, thebispecific HER2/EGFR is lapatinib/GW572016.

Additional antibody-based inhibitors can be raised according to knownmethods by administering the appropriate antigen or epitope to a hostanimal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep,and mice, among others. Various adjuvants known in the art can be usedto enhance antibody production.

Although antibodies useful in practicing the invention can bepolyclonal, monoclonal antibodies are preferred. Monoclonal antibodiescan be prepared and isolated using any technique that provides for theproduction of antibody molecules by continuous cell lines in culture.Techniques for production and isolation include but are not limited tothe hybridoma technique originally described by Kohler and Milstein(Nature, 1975, 256: 495-497); the human B-cell hybridoma technique(Kosbor et al., 1983, Immunology Today 4:72; Cote et al., 1983, Proc.Natl. Acad. Sci. USA 80: 2026-2030); and the EBV-hybridoma technique(Cole et al, 1985, Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., pp. 77-96).

Alternatively, techniques described for the production of single chainantibodies (see, e.g., U.S. Pat. No. 4,946,778) can be adapted toproduce single chain antibodies with desired specificity. Antibody-basedinhibitors useful in practicing the present invention also includeantibody fragments including but not limited to F(ab′).sub.2 fragments,which can be generated by pepsin digestion of an intact antibodymolecule, and Fab fragments, which can be generated by reducing thedisulfide bridges of the F(ab′).sub.2 fragments. Alternatively, Faband/or scFv expression libraries can be constructed (see, e.g., Huse etal., 1989, Science 246: 1275-1281) to allow rapid identification offragments having the desired specificity.

Techniques for the production and isolation of monoclonal antibodies andantibody fragments are well-known in the art, and are described inHarlow and Lane, 1988, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, and in J. W. Goding, 1986, Monoclonal Antibodies:Principles and Practice, Academic Press, London. Humanized anti-EGFRantibodies and antibody fragments can also be prepared according toknown techniques such as those described in Vaughn, T. J. et al., 1998,Nature Biotech. 16:535-539 and references cited therein, and suchantibodies or fragments thereof are also useful in practicing thepresent invention.

Inhibitors for use in the present invention can alternatively be basedon antisense oligonucleotide constructs. Anti-sense oligonucleotides,including anti-sense RNA molecules and anti-sense DNA molecules, wouldact to directly block the translation of target mRNA by binding theretoand thus preventing protein translation or increasing mRNA degradation,thus decreasing the level of the target protein, and thus activity, in acell. For example, antisense oligonucleotides of at least about 15 basesand complementary to unique regions of the mRNA transcript sequenceencoding EGFR or HER2 can be synthesized, e.g., by conventionalphosphodiester techniques and administered by e.g., intravenousinjection or infusion. Methods for using antisense techniques forspecifically inhibiting gene expression of genes whose sequence is knownare well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131;6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732).

Small inhibitory RNAs (siRNAs) can also function as inhibitors for usein the present invention. Target gene expression can be reduced bycontacting the tumor, subject or cell with a small double stranded RNA(dsRNA), or a vector or construct causing the production of a smalldouble stranded RNA, such that expression of the target gene isspecifically inhibited (i.e. RNA interference or RNAi). Methods forselecting an appropriate dsRNA or dsRNA-encoding vector are well knownin the art for genes whose sequence is known (e.g. see Tuschi, T., etal. (1999) Genes Dev. 13(24):3191-3197; Elbashir, S. M. et al. (2001)Nature 411:494-498; Hannon, G. J. (2002) Nature 418:244-251; McManus, M.T. and Sharp, P. A. (2002) Nature Reviews Genetics 3:737-747;Bremmelkamp, T. R. et al. (2002) Science 296:550-553; U.S. Pat. Nos.6,573,099 and 6,506,559; and International Patent Publication Nos. WO01/36646, WO 99/32619, and WO 01/68836).

Ribozymes can also function as inhibitors for use in the presentinvention. Ribozymes are enzymatic RNA molecules capable of catalyzingthe specific cleavage of RNA. The mechanism of ribozyme action involvessequence specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage.Engineered hairpin or hammerhead motif ribozyme molecules thatspecifically and efficiently catalyze endonucleolytic cleavage of mRNAsequences are thereby useful within the scope of the present invention.Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites, which typically include the following sequences, GUA,GUU, and GUC. Once identified, short RNA sequences of between about 15and 20 ribonucleotides corresponding to the region of the target genecontaining the cleavage site can be evaluated for predicted structuralfeatures, such as secondary structure, that can render theoligonucleotide sequence unsuitable. The suitability of candidatetargets can also be evaluated by testing their accessibility tohybridization with complementary oligonucleotides, using, e.g.,ribonuclease protection assays.

Both antisense oligonucleotides and ribozymes useful as inhibitors canbe prepared by known methods. These include techniques for chemicalsynthesis such as, e.g., by solid phase phosphoramadite chemicalsynthesis. Alternatively, anti-sense RNA molecules can be generated byin vitro or in vivo transcription of DNA sequences encoding the RNAmolecule. Such DNA sequences can be incorporated into a wide variety ofvectors that incorporate suitable RNA polymerase promoters such as theT7 or SP6 polymerase promoters. Various modifications to theoligonucleotides of the invention can be introduced as a means ofincreasing intracellular stability and half-life. Possible modificationsinclude but are not limited to the addition of flanking sequences ofribonucleotides or deoxyribonucleotides to the 5′ and/or 3′ ends of themolecule, or the use of phosphorothioate or 2′-O-methyl rather thanphosphodiesterase linkages within the oligonucleotide backbone.

In the context of the methods of treatment of this invention, inhibitors(such as an EGFR inhibitor or a HER2 inhibitor) are used as acomposition comprised of a pharmaceutically acceptable carrier and anon-toxic therapeutically effective amount of an EGFR kinase inhibitorcompound (including pharmaceutically acceptable salts thereof).

The term “pharmaceutically acceptable salts” refers to salts preparedfrom pharmaceutically acceptable non-toxic bases or acids. When acompound of the present invention is acidic, its corresponding salt canbe conveniently prepared from pharmaceutically acceptable non-toxicbases, including inorganic bases and organic bases. Salts derived fromsuch inorganic bases include aluminum, ammonium, calcium, copper (cupricand cuprous), ferric, ferrous, lithium, magnesium, manganese (manganicand manganous), potassium, sodium, zinc and the like salts. Particularlypreferred are the ammonium, calcium, magnesium, potassium and sodiumsalts. Salts derived from pharmaceutically acceptable organic non-toxicbases include salts of primary, secondary, and tertiary amines, as wellas cyclic amines and substituted amines such as naturally occurring andsynthesized substituted amines. Other pharmaceutically acceptableorganic non-toxic bases from which salts can be formed include ionexchange resins such as, for example, arginine, betaine, caffeine,choline, N′,N′-dibenzylethylenediamine, diethylamine,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperidine, polyamine resins,procaine, purines, theobromine, triethylameine, trimethylamine,tripropylamine, tromethamine and the like.

When a compound used in the present invention is basic, itscorresponding salt can be conveniently prepared from pharmaceuticallyacceptable non-toxic acids, including inorganic and organic acids. Suchacids include, for example, acetic, benzenesulfonic, benzoic,camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic,hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic,methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric,succinic, sulfuric, tartaric, p-toluenesulfonic acid and the like.Particularly preferred are citric, hydrobromic, hydrochloric, maleic,phosphoric, sulfuric and tartaric acids.

Pharmaceutical compositions used in the present invention comprising aninhibitor compound (including pharmaceutically acceptable salts thereof)as active ingredient, can include a pharmaceutically acceptable carrierand optionally other therapeutic ingredients or adjuvants. Othertherapeutic agents may include those cytotoxic, chemotherapeutic oranti-cancer agents, or agents which enhance the effects of such agents,as listed above. The compositions include compositions suitable fororal, rectal, topical, and parenteral (including subcutaneous,intramuscular, and intravenous) administration, although the mostsuitable route in any given case will depend on the particular host, andnature and severity of the conditions for which the active ingredient isbeing administered. The pharmaceutical compositions may be convenientlypresented in unit dosage form and prepared by any of the methods wellknown in the art of pharmacy

In practice, the inhibitor compounds (including pharmaceuticallyacceptable salts thereof) of this invention can be combined as theactive ingredient in intimate admixture with a pharmaceutical carrieraccording to conventional pharmaceutical compounding techniques. Thecarrier may take a wide variety of forms depending on the form ofpreparation desired for administration, e.g. oral or parenteral(including intravenous). Thus, the pharmaceutical compositions of thepresent invention can be presented as discrete units suitable for oraladministration such as capsules, cachets or tablets each containing apredetermined amount of the active ingredient. Further, the compositionscan be presented as a powder, as granules, as a solution, as asuspension in an aqueous liquid, as a non-aqueous liquid, as anoil-in-water emulsion, or as a water-in-oil liquid emulsion. In additionto the common dosage forms set out above, an inhibitor compound(including pharmaceutically acceptable salts of each component thereof)may also be administered by controlled release means and/or deliverydevices. The combination compositions may be prepared by any of themethods of pharmacy. In general, such methods include a step of bringinginto association the active ingredients with the carrier thatconstitutes one or more necessary ingredients. In general, thecompositions are prepared by uniformly and intimately admixing theactive ingredient with liquid carriers or finely divided solid carriersor both. The product can then be conveniently shaped into the desiredpresentation.

An inhibitor compound (including pharmaceutically acceptable saltsthereof) used in this invention, can also be included in pharmaceuticalcompositions in combination with one or more other therapeuticallyactive compounds. Other therapeutically active compounds may includethose cytotoxic, chemotherapeutic or anti-cancer agents, or agents whichenhance the effects of such agents, as listed above.

Thus in one embodiment of this invention, the pharmaceutical compositioncan comprise an inhibitor compound in combination with an anticanceragent, wherein the anti-cancer agent is a member selected from the groupconsisting of alkylating drugs, antimetabolites, microtubule inhibitors,podophyllotoxins, antibiotics, nitrosoureas, hormone therapies, kinaseinhibitors, activators of tumor cell apoptosis, and antiangiogenicagents.

The pharmaceutical carrier employed can be, for example, a solid,liquid, or gas. Examples of solid carriers include lactose, terra alba,sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, andstearic acid. Examples of liquid carriers are sugar syrup, peanut oil,olive oil, and water. Examples of gaseous carriers include carbondioxide and nitrogen.

In preparing the compositions for oral dosage form, any convenientpharmaceutical media may be employed. For example, water, glycols, oils,alcohols, flavoring agents, preservatives, coloring agents, and the likemay be used to form oral liquid preparations such as suspensions,elixirs and solutions; while carriers such as starches, sugars,microcrystalline cellulose, diluents, granulating agents, lubricants,binders, disintegrating agents, and the like may be used to form oralsolid preparations such as powders, capsules and tablets. Because oftheir ease of administration, tablets and capsules are the preferredoral dosage units whereby solid pharmaceutical carriers are employed.Optionally, tablets may be coated by standard aqueous or nonaqueoustechniques.

A tablet containing the composition used for this invention may beprepared by compression or molding, optionally with one or moreaccessory ingredients or adjuvants. Compressed tablets may be preparedby compressing, in a suitable machine, the active ingredient in afree-flowing form such as powder or granules, optionally mixed with abinder, lubricant, inert diluent, surface active or dispersing agent.Molded tablets may be made by molding in a suitable machine, a mixtureof the powdered compound moistened with an inert liquid diluent. Eachtablet preferably contains from about 0.05 mg to about 5 g of the activeingredient and each cachet or capsule preferably contains from about0.05 mg to about 5 g of the active ingredient.

For example, a formulation intended for the oral administration tohumans may contain from about 0.5 mg to about 5 g of active agent,compounded with an appropriate and convenient amount of carrier materialthat may vary from about 5 to about 95 percent of the total composition.Unit dosage forms will generally contain between from about 1 mg toabout 2 g of the active ingredient, typically 25 mg, 50 mg, 100 mg, 200mg, 300 mg, 400 mg, 500 mg, 600 mg, 800 mg, or 1000 mg.

Pharmaceutical compositions used in the present invention suitable forparenteral administration may be prepared as solutions or suspensions ofthe active compounds in water. A suitable surfactant can be includedsuch as, for example, hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofin oils. Further, a preservative can be included to prevent thedetrimental growth of microorganisms.

Pharmaceutical compositions used in the present invention suitable forinjectable use include sterile aqueous solutions or dispersions.Furthermore, the compositions can be in the form of sterile powders forthe extemporaneous preparation of such sterile injectable solutions ordispersions. In all cases, the final injectable form must be sterile andmust be effectively fluid for easy syringability. The pharmaceuticalcompositions must be stable under the conditions of manufacture andstorage; thus, preferably should be preserved against the contaminatingaction of microorganisms such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (e.g., glycerol, propylene glycol and liquid polyethyleneglycol), vegetable oils, and suitable mixtures thereof.

Pharmaceutical compositions for the present invention can be in a formsuitable for topical sue such as, for example, an aerosol, cream,ointment, lotion, dusting powder, or the like. Further, the compositionscan be in a form suitable for use in transdermal devices. Theseformulations may be prepared, utilizing an inhibitor compound (includingpharmaceutically acceptable salts thereof), via conventional processingmethods. As an example, a cream or ointment is prepared by admixinghydrophilic material and water, together with about 5 wt % to about 10wt % of the compound, to produce a cream or ointment having a desiredconsistency.

Pharmaceutical compositions for this invention can be in a form suitablefor rectal administration wherein the carrier is a solid. It ispreferable that the mixture forms unit dose suppositories. Suitablecarriers include cocoa butter and other materials commonly used in theart. The suppositories may be conveniently formed by first admixing thecomposition with the softened or melted carrier(s) followed by chillingand shaping in molds.

In addition to the aforementioned carrier ingredients, thepharmaceutical formulations described above may include, as appropriate,one or more additional carrier ingredients such as diluents, buffers,flavoring agents, binders, surface-active agents, thickeners,lubricants, preservatives (including anti-oxidants) and the like.Furthermore, other adjuvants can be included to render the formulationisotonic with the blood of the intended recipient. Compositionscontaining an inhibitor compound (including pharmaceutically acceptablesalts thereof) may also be prepared in powder or liquid concentrateform.

Dosage levels for the compounds used for practicing this invention willbe approximately as described herein, or as described in the art forthese compounds. It is understood, however, that the specific dose levelfor any particular patient will depend upon a variety of factorsincluding the age, body weight, general health, sex, diet, time ofadministration, route of administration, rate of excretion, drugcombination and the severity of the particular disease undergoingtherapy.

Many alternative experimental methods known in the art may besuccessfully substituted for those specifically described herein in thepractice of this invention, as for example described in many of theexcellent manuals and textbooks available in the areas of technologyrelevant to this invention (e.g. Using Antibodies, A Laboratory Manual,edited by Harlow, E. and Lane, D., 1999, Cold Spring Harbor LaboratoryPress, (e.g. ISBN 0-87969-544-7); Roe B. A. et. al. 1996, DNA Isolationand Sequencing (Essential Techniques Series), John Wiley & Sons. (e.g.ISBN 0-471-97324-0); Methods in Enzymology: Chimeric Genes andProteins”, 2000, ed. J. Abelson, M. Simon, S. Emr, J. Thorner. AcademicPress; Molecular Cloning: a Laboratory Manual, 2001, 3rd Edition, byJoseph Sambrook and Peter MacCallum, (the former Maniatis Cloningmanual) (e.g. ISBN 0-87969-577-3); Current Protocols in MolecularBiology, Ed. Fred M. Ausubel, et. al. John Wiley & Sons (e.g. ISBN0-471-50338-X); Current Protocols in Protein Science, Ed. John E.Coligan, John Wiley & Sons (e.g. ISBN 0-471-11184-8); and Methods inEnzymology: Guide to protein Purification, 1990, Vol. 182, Ed.Deutscher, M. P., Academic Press, Inc. (e.g. ISBN 0-12-213585-7)), or asdescribed in the many university and commercial websites devoted todescribing experimental methods in molecular biology.

It will be appreciated by one of skill in the medical arts that theexact manner of administering to the patient of a therapeuticallyeffective amount of an inhibitor as described herein (for example anEGFR kinase inhibitor, bispecific EGFR kinase inhibitor, or HER2inhibitor) following a diagnosis of a patient's likely responsiveness tothe inhibitor will be at the discretion of the attending physician. Themode of administration, including dosage, combination with otheranti-cancer agents, timing and frequency of administration, and thelike, may be affected by the diagnosis of a patient's likelyresponsiveness to the inhibitor, as well as the patient's condition andhistory. Thus, even patients diagnosed with tumors predicted to berelatively insensitive to the type of inhibitor may still benefit fromtreatment with such inhibitor, particularly in combination with otheranti-cancer agents, or agents that may alter a tumor's sensitivity tothe inhibitor.

For purposes of the present invention, “co-administration of” and“co-administering” an inhibitor with an additional anti-cancer agent(both components referred to hereinafter as the “two active agents”)refer to any administration of the two active agents, either separatelyor together, where the two active agents are administered as part of anappropriate dose regimen designed to obtain the benefit of thecombination therapy. Thus, the two active agents can be administeredeither as part of the same pharmaceutical composition or in separatepharmaceutical compositions. The additional agent can be administeredprior to, at the same time as, or subsequent to administration of theinhibitor, or in some combination thereof. Where the inhibitor isadministered to the patient at repeated intervals, e.g., during astandard course of treatment, the additional agent can be administeredprior to, at the same time as, or subsequent to, each administration ofthe inhibitor, or some combination thereof, or at different intervals inrelation to the inhibitor treatment, or in a single dose prior to, atany time during, or subsequent to the course of treatment with theinhibitor.

The inhibitor will typically be administered to the patient in a doseregimen that provides for the most effective treatment of the cancer(from both efficacy and safety perspectives) for which the patient isbeing treated, as known in the art, and as disclosed, e.g. inInternational Patent Publication No. WO 01/34574. In conducting thetreatment method of the present invention, the inhibitor can beadministered in any effective manner known in the art, such as by oral,topical, intravenous, intra-peritoneal, intramuscular, intra-articular,subcutaneous, intranasal, intra-ocular, vaginal, rectal, or intradermalroutes, depending upon the type of cancer being treated, the type ofinhibitor being used (for example, small molecule, antibody, RNAi,ribozyme or antisense construct), and the medical judgement of theprescribing physician as based, e.g., on the results of publishedclinical studies.

The amount of inhibitor administered and the timing of inhibitoradministration will depend on the type (species, gender, age, weight,etc.) and condition of the patient being treated, the severity of thedisease or condition being treated, and on the route of administration.For example, small molecule inhibitors can be administered to a patientin doses ranging from 0.001 to 100 mg/kg of body weight per day or perweek in single or divided doses, or by continuous infusion (see forexample, International Patent Publication No. WO 01/34574). Inparticular, erlotinib HCl can be administered to a patient in dosesranging from 5-200 mg per day, or 100-1600 mg per week, in single ordivided doses, or by continuous infusion. A preferred dose is 150mg/day. Antibody-based inhibitors, or antisense, RNAi or ribozymeconstructs, can be administered to a patient in doses ranging from 0.1to 100 mg/kg of body weight per day or per week in single or divideddoses, or by continuous infusion. In some instances, dosage levels belowthe lower limit of the aforethe range may be more than adequate, whilein other cases still larger doses may be employed without causing anyharmful side effect, provided that such larger doses are first dividedinto several small doses for administration throughout the day.

The inhibitors and other additional agents can be administered eitherseparately or together by the same or different routes, and in a widevariety of different dosage forms. For example, the inhibitor ispreferably administered orally or parenterally. Where the inhibitor iserlotinib HCl (TARCEVA™), oral administration is preferable. Both theinhibitor and other additional agents can be administered in single ormultiple doses.

The inhibitor can be administered with various pharmaceuticallyacceptable inert carriers in the form of tablets, capsules, lozenges,troches, hard candies, powders, sprays, creams, salves, suppositories,jellies, gels, pastes, lotions, ointments, elixirs, syrups, and thelike. Administration of such dosage forms can be carried out in singleor multiple doses. Carriers include solid diluents or fillers, sterileaqueous media and various non-toxic organic solvents, etc. Oralpharmaceutical compositions can be suitably sweetened and/or flavored.

The inhibitor can be combined together with various pharmaceuticallyacceptable inert carriers in the form of sprays, creams, salves,suppositories, jellies, gels, pastes, lotions, ointments, and the like.Administration of such dosage forms can be carried out in single ormultiple doses. Carriers include solid diluents or fillers, sterileaqueous media, and various non-toxic organic solvents, etc.

All formulations comprising proteinaceous inhibitors should be selectedso as to avoid denaturation and/or degradation and loss of biologicalactivity of the inhibitor.

Methods of preparing pharmaceutical compositions comprising an inhibitorare known in the art, and are described, e.g. in International PatentPublication No. WO 01/34574. In view of the teaching of the presentinvention, methods of preparing pharmaceutical compositions comprisingan inhibitor will be apparent from the above-cited publications and fromother known references, such as Remington's Pharmaceutical Sciences,Mack Publishing Company, Easton, Pa., 18^(th) edition (1990).

For oral administration of inhibitors, tablets containing one or both ofthe active agents are combined with any of various excipients such as,for example, micro-crystalline cellulose, sodium citrate, calciumcarbonate, dicalcium phosphate and glycine, along with variousdisintegrants such as starch (and preferably corn, potato or tapiocastarch), alginic acid and certain complex silicates, together withgranulation binders like polyvinyl pyrrolidone, sucrose, gelatin andacacia. Additionally, lubricating agents such as magnesium stearate,sodium lauryl sulfate and talc are often very useful for tabletingpurposes. Solid compositions of a similar type may also be employed asfillers in gelatin capsules; preferred materials in this connection alsoinclude lactose or milk sugar as well as high molecular weightpolyethylene glycols. When aqueous suspensions and/or elixirs aredesired for oral administration, the inhibitor may be combined withvarious sweetening or flavoring agents, coloring matter or dyes, and, ifso desired, emulsifying and/or suspending agents as well, together withsuch diluents as water, ethanol, propylene glycol, glycerin and variouslike combinations thereof.

For parenteral administration of either or both of the active agents,solutions in either sesame or peanut oil or in aqueous propylene glycolmay be employed, as well as sterile aqueous solutions comprising theactive agent or a corresponding water-soluble salt thereof. Such sterileaqueous solutions are preferably suitably buffered, and are alsopreferably rendered isotonic, e.g., with sufficient saline or glucose.These particular aqueous solutions are especially suitable forintravenous, intramuscular, subcutaneous and intraperitoneal injectionpurposes. The oily solutions are suitable for intra-articular,intramuscular and subcutaneous injection purposes. The preparation ofall these solutions under sterile conditions is readily accomplished bystandard pharmaceutical techniques well known to those skilled in theart. Any parenteral formulation selected for administration ofproteinaceous inhibitors should be selected so as to avoid denaturationand loss of biological activity of the inhibitor.

Additionally, it is possible to topically administer either or both ofthe active agents, by way of, for example, creams, lotions, jellies,gels, pastes, ointments, salves and the like, in accordance withstandard pharmaceutical practice. For example, a topical formulationcomprising an inhibitor in about 0.1% (w/v) to about 5% (w/v)concentration can be prepared.

For veterinary purposes, the active agents can be administeredseparately or together to animals using any of the forms and by any ofthe routes described above. In a preferred embodiment, the inhibitor isadministered in the form of a capsule, bolus, tablet, liquid drench, byinjection or as an implant. As an alternative, the inhibitor can beadministered with the animal feedstuff, and for this purpose aconcentrated feed additive or premix may be prepared for a normal animalfeed. Such formulations are prepared in a conventional manner inaccordance with standard veterinary practice.

One of skill in the medical arts, particularly pertaining to theapplication of diagnostic tests and treatment with therapeutics, willrecognize that biological systems may exhibit variability and may notalways be entirely predictable, and thus many good diagnostic tests ortherapeutics are occasionally ineffective. Thus, it is ultimately up tothe judgement of the attending physician to determine the mostappropriate course of treatment for an individual patient, based upontest results, patient condition and history, and his own experience.There may even be occasions, for example, when a physician will chooseto treat a patient with an EGFR inhibitor even when a tumor is notpredicted to be particularly sensitive to EGFR kinase inhibitors, basedon data from diagnostic tests or from other criteria, particularly ifall or most of the other obvious treatment options have failed, or ifsome synergy is anticipated when given with another treatment. The factthat the EGFR inhibitors as a class of drugs are relatively welltolerated compared to many other anti-cancer drugs, such as moretraditional chemotherapy or cytotoxic agents used in the treatment ofcancer, makes this a more viable option.

Methods of Advertising

The invention herein also encompasses a method for advertising an EGFRor HER2 inhibitor, or a pharmaceutically acceptable composition thereofcomprising promoting, to a target audience, the use of the inhibitor orpharmaceutical composition thereof for treating a patient populationwith a type of cancer which is characterized by ERBB2 hypomethylation.

Advertising is generally paid communication through a non-personalmedium in which the sponsor is identified and the message is controlled.Advertising for purposes herein includes publicity, public relations,product placement, sponsorship, underwriting, and sales promotion. Thisterm also includes sponsored informational public notices appearing inany of the print communications media designed to appeal to a massaudience to persuade, inform, promote, motivate, or otherwise modifybehavior toward a favorable pattern of purchasing, supporting, orapproving the invention herein.

The advertising and promotion of the diagnostic method herein may beaccomplished by any means. Examples of advertising media used to deliverthese messages include television, radio, movies, magazines, newspapers,the internet, and billboards, including commercials, which are messagesappearing in the broadcast media. Advertisements also include those onthe seats of grocery carts, on the walls of an airport walkway, and onthe sides of buses, or heard in telephone hold messages or in-store PAsystems, or anywhere a visual or audible communication can be placed.

More specific examples of promotion or advertising means includetelevision, radio, movies, the internet such as webcasts and webinars,interactive computer networks intended to reach simultaneous users,fixed or electronic billboards and other public signs, posters,traditional or electronic literature such as magazines and newspapers,other media outlets, presentations or individual contacts by, e.g.,e-mail, phone, instant message, postal, courier, mass, or carrier mail,in-person visits, etc.

The type of advertising used will depend on many factors, for example,on the nature of the target audience to be reached, e.g., hospitals,insurance companies, clinics, doctors, nurses, and patients, as well ascost considerations and the relevant jurisdictional laws and regulationsgoverning advertising of medicaments and diagnostics. The advertisingmay be individualized or customized based on user characterizationsdefined by service interaction and/or other data such as userdemographics and geographical location.

This invention will be better understood from the Examples that follow.However, one skilled in the art will readily appreciate that thespecific methods and results discussed are merely illustrative of theinvention as described more fully in the claims which follow thereafter,and are not to be considered in any way limited thereto.

All patents, published patent applications and other referencesdisclosed herein are hereby expressly incorporated by reference in theirentirety.

III. Examples Example 1 Materials and Methods

Cell Lines:

All of the NSCLC cell lines were purchased from the American Type CellCulture Collection (ATCC) or were provided by Adi Gazdar and John Minnaat UT Southwestern. The immortalized bronchial epithelial (gBECs) andsmall airway (gSACs) cell lines were created at Genentech using atricistronic vector containing cdk4, hTERT, and G418 as a selectionmarker. The tricistronic vector was engineered from the pQCXIN backbonecontaining hTERT. The immortalization process was based on previouslypublished protocols with some modification (Ramirez et al., 2004, CancerRes 64:9027; Sato et al., 2006, Cancer Res 66:2116). The gBECs and gSACshave a diploid karyotype and are non-tumorigenic.

Tumor Samples:

DNA methylation assays were performed on tumor DNA isolated from FFPEbiopsy material obtained from patients enrolled in TRIBUTE, a phase IIItrial sponsored by Genentech to compare the survival of 1,079 stage IIIBor stage IV NSCLC patients who received Erlotinib administeredconcurrently with a regimen of carboplatin and paclitaxel (n=539) topatients who received carboplatin and paclitaxel alone (n=540) (Yauch etal., 2005, Clin Cancer Res 11:8686). DNA was available for 343 TRIBUTEpatients and 112 MetMAb patients.

Demethylation of Genomic DNA:

Cells to used for negative controls in the methylation assays were grownin RPMI 1640 supplemented with 10% fetal bovine serum and 2 mML-Glutamine Cells were seeded on day 0 at 4000-9000 cells/cm2 and dosedwith 1 μM 5-aza-2′-deoxycytidine (SIGMA-ALDRICH Cat No. A3656) or DMSOcontrol (Cat No. D2650) on days 1, 3, and 5. On day 6 cells were washedonce in cold Phosphate Buffered Saline and harvested by scraping inTrizol (Invitrogen, Cat No 15596018) and extracted for RNA or flashfrozen for later RNA extraction.

Illumina Infinium Methylation Analysis:

1 μg of genomic DNA from each of 96 NSCLC cell lines wasbisulfite-converted and analyzed on the Illumina Infinium 450Kmethylation. Methylation data were processed using the Bioconductor lumisoftware package (Du et al., 2008, Bioinformatics 24:1547). The Infinium450K platform includes Infinium I and II assays on the same array. TheInfinium I assay employs two bead types per CpG locus, with themethylated state reported by the red dye in some cases and the green dyein others (identical to the previous Infinium 27K platform). TheInfinium II assay uses one bead type and always reports the methylatedstate with the same dye, making dye bias a concern. After discarding onearray with high background signal, a two-stage normalization procedurewas applied to the remaining arrays. First, for each array, a color-biascorrection curve was estimated from Infinium I data using a smoothquantile normalization method; this correction curve was then applied toall data from that array. Second, arrays were normalized to one anotherby applying standard quantile normalization to all color-correctedsignals.

After pre-processing, both methylation M-values (log₂ ratios ofmethylated to unmethylated probes) and β-values (a resealing of theM-values to the 0 and 1 range via logistic transform) were computed foreach sample (Du et al, 2010, BMC Bioinformatics 11:587). Forvisualization, agglomerative hierarchical clustering of β-values wasperformed using complete linkage and Euclidean distance. DMRs wereidentified by first computing a moving average for each cell line'sM-values (500 bp windows centered on interrogated CpG sites); then, at-test was used to contrast the window scores associated with a trainingset of randomly selected 10 Epithelial-like and 10 Mesenchymal-likelines. DMR p-values were adjusted to control the False Discovery rate(Benjamini and Hochberg, 1995) and compared to a cutoff of 0.01. Toenrich for more biologically relevant phenomena, downstream analysesonly considered those differentially methylated regions whose averagewindow scores (i) differed by at least 2 between the sensitive andresistant lines, and (ii) had opposite sign in the two sets of celllines. Finally, contiguous DMRs which met all of these criteria weremerged into a single DMR if they were separated by less than 2 kb.

Bisulfite Sequencing and Analysis:

To confirm DNA methylation status of candidate genes, 2 μg genomic DNAwas bisulfite-converted using the EZ DNA Methylation-Gold kit (ZymoResearch). Primers specific to the converted DNA were designed usingMethyl Primer Express software v1.0 (Applied Biosystems). PCRamplification was performed with 1 μl of bisulfite-converted DNA in a25-μl reaction using Platinum PCR supermix (Invitrogen). The PCRthermocycling conditions were as follows: 1 initial denaturation cycleof 95° C. for 10 minutes, followed by 10 cycles of 94° C. for 30seconds, 65° C. for 1 minute and decreasing by 1° C. every cycle, and72° C. for 1 minute, followed by 30 cycles of 94° C. for 30 seconds, 55°C. for 1.5 minutes, and 72° C. for 1 minute, followed by a finalextension at 72° C. for 15 minutes. PCR products were resolved byelectrophoresis using 2% agarose E-gels containing ethidium bromide(Invitrogen) and visualized using a Fluor Chem 8900 camera (AlphaInnotech).

PCR products were ligated into the pCR4-TOPO vector using the TOPO TACloning kit (Invitrogen) according to the manufacturer's instructions. 2μl of ligated plasmid DNA were transformed into TOP10 competent bacteria(Invitrogen), and 100 μl transformed bacteria were plated on LB-agarplates containing 50 μg/ml carbenicillin (Teknova) and incubatedovernight at 37° C. Twelve colonies per cell line for each candidatelocus were inoculated into 1 ml of LB containing 50 μg/ml carbenicillinand grown overnight in a shaking incubator at 37° C. Plasmid DNA wasisolated using a Qiaprep miniprep kit in 96-well format (Qiagen) andsequenced on a 3730×1 DNA Analyzer (Applied Biosystems). Sequencing datawere analyzed using Sequencher v4.5 software and BiQ Analyzer software.Bisulfite-converted sequences were first aligned and trimmed toreference sequences for each candidate locus using Sequencher toevaluate sequence quality and confirm cytosine conversion during sodiumbisulfite treatment. Trimmed sequences were then evaluated formethylation status at individual CpG sites using BiQ Analyzer software.

Pyrosequencing:

Bisulfite-specific PCR (BSP) primers were designed using Methyl PrimerExpress software v1.0 (Applied Biosystems) or PyroMark Assay Designsoftware v2.0 (Qiagen). PCR primers were synthesized with a 5′ biotinlabel on either the forward or reverse primer to facilitate binding ofthe PCR product to Streptavidin Sepharose beads. Sequencing primers weredesigned in the reverse direction of the 5′-biotin-labeled PCR primerusing PyroMark Assay Design software v2.0 (Qiagen). 1 μl bisulfitemodified DNA was amplified in a 25 μl reaction using Platinum PCRSupermix (Invitrogen) and 20 μl of PCR product was used for sequencingon the Pyromark Q24 (Qiagen). PCR products were incubated withStreptavidin Sepharose beads for 10 minutes followed by washes with 70%ethanol, Pyromark denaturation solution, and Pyromark wash buffer.Denatured PCR products were then sequenced using 0.3 μM sequencingprimer. Pyrograms were visualized and evaluated for sequence quality,and percent methylation at individual CpG sites was determined usingPyroMark software version 2.0.4 (Qiagen). The following primers areexemplary primers used in the ERBB2 pyrosequencing assays:

ERBB2 Pyrosequencing Primers: (SEQ ID NO: 3)1 Forward: 5'-GGTTTAAGTGGGTTAGGTGTG-3' (SEQ ID NO: 4)1 Reverse, biotin: 5'-CAATTATAAACATCTAAACCCAAACTAC A-3' (SEQ ID NO: 5)1 Sequencing: 5'-AGT TTTATGTTTTATGGT TGA-3' (SEQ ID NO: 6)Nested Forward: 5'-TAGTTTTATGTTTTATGGTTGATGGTT-3' (SEQ ID NO: 7)Nested Reverse, biotin: 5'-CCAAAACCAACTAACAAAATATA TACC-3'(SEQ ID NO: 8) Nested Sequencing: 5'-TTGGGTAGGTATGTAGG-3'

Promoter Enhancer Activity Luciferase Reporter Assay:

Promoter enhancer activity of a differentially methylated region(identified by Infinium array profiling) of the ERBB2 gene was assessedusing Dual-Luciferase Reporter Assay System (Promega). A 1791-bp regionwithin the first intron of ERBB2 was cloned into the pGL4 luciferasereporter vector according to the manufacturer's instructions. Cells weretransfected with the control promoter plus ERBB2 putative enhancerregion, and luciferase activity was measured using a standardluminometer at 24, 48, and 72 hour time points following transfection.

Quantitative Methylation Specific PCR:

Quantitative methylation specific PCR (qMSP) assays were designed usinggenetic loci identified in our candidate screen as differentiallymethylated in erlotinib-sensitive and resistant NSCLC cell lines. Aminimum of 10 ng of sodium bisulfite converted DNA was amplified withvarious 20× Custom Taqman Gene Expression Assays, Applied Biosystems,Cat No. 4331348) using TaqMan® Universal PCR Master Mix, No AmpErase®UNG (Applied Biosystems, Cat No. 4324018) with cycling conditions of 95°C. 10 min, then 50 cycles of 95° C. for 15 sec and 60° C. for 1 min.Amplification was done on a 7900HT system and analyzed using SDSsoftware (Applied Biosystems). DNA content was normalized using meRNasePTaqman assay.

Pre-Amplification of FFPE Clinical Trial Material:

A pre-amplification method for methylation analysis of pg amounts of DNAextracted from formalin-fixed paraffin embedded (FFPE) tissue wasdeveloped. 2 μl (equivalent of 10 pg-1 ng) bisulfite converted DNA wasfirst amplified in a 20 μl reaction with 0.1×qMSP primer-probeconcentrations using TaqMan® Universal PCR Master Mix, No AmpErase® UNG(Applied Biosystems, Cat No. 4324018) and cycling conditions of 95° C.10 min, then 14 cycles of 95° C. for 15 sec and 60° C. for 1 min. 1 μlof the pre-amplified material was then amplified in a second PCRreaction with cycling conditions of 95° C. 10 min, then 50 cycles of 95°C. for 15 sec and 60° C. for 1 min. DNA content was confirmed using apre-amplification with the reference meRNaseP Taqman assay and onlysamples that were positive for meRNaseP were included in furtheranalysis of qMSP reactions. All reactions were performed in duplicate.

Example 2 Hypomethylation of the ERBB2 DMR Correlates with anEpithelial-Like Phenotype in NSCLC Cell Lines and in NSCLC PrimaryTumors

A CpG site near exon 4 of the ERBB2 proto-oncogene was identified as adifferentially methylated region (DMR), based on methylation profilingof epithelial and meschenchymal-like NSCLC cell lines. Methylationprofiling was performed using Infinium Methylation Analysis, andverification of the methylation status was verified by direct sequencingof cloned fragments of sodium bisulfite-DNA.

Pyrosequencing was used to determine quantitative methylation status ofthe DMR in NSCLC primary tumors and matched normal tissues. Quantitativemethylation was determined at 6 consecutive CpG sites by Pyromarkanalysis software using the equation % methylation=(C peak height×100/Cpeak height+T peak height, FIG. 2 (showing the mean percent methylationof 6 individual CpG sites, with a P-value p<0.06 determined using aStudent's t-test). As shown in FIG. 2, this intragenic DMR of ERBB2appeared to be hypomethylated relative to normal adjacent tissue.

In silico analysis of this region using the UCSC genome browsersuggested that the differentially methylated CpG site corresponding toprobe cg00459816 (Type II Illumina Infinium 450K methylation arrayprobe, representating a single CpG site at chromosomal coordinates: NCBIbuild 36/hg18 chr17:35115639 (Illumina, Inc., San Diego, Calif.)overlapped with a potential regulatory element. Because this region wasnot within a CpG island and was not particularly GC rich, pyrosequencingprimers flanking this region were designed to determine its methylationstatus in a panel of epithelial-like and mesenchymal-like cell lines. Apattern of hypomethylation (mean methylation of 6 CpG sites ≦20%) in 13of 16 epithelial-like lines relative to mesenchymal-like lines (meanmethylation ≧70% in 20 of 21 mesenchymal-like lines; P<0.001) wasobserved. FIG. 3 shows the results of this analysis, with data beingrepresented as mean+/−SD percentage of methylation at 6 consecutive CpGsites in the sequenced region.

Only one mesenchymal-like line, H1435, was hypomethylated at this locus.This exception was not surprising given our previous observation thatH1435 was identified as a mesenchymal-like line by EMT expressionanalysis.

Example 3 Hypomethylation of the ERBB2 DMR Correlates with ERBB2Expression

The relative expression level of ERBB2 mRNA in the NSCLC cell lines wasdetermined using TaqMan-based Fluidigm gene expression analysis. Asshown in FIG. 4, epithelial-like lines exhibited significantly higherlevels of ERBB2 expression (P<0.001) than mesenchymal-like lines. Thefinding that hypomethylation of the ERBB2 locus is highly correlatedwith both higher expression of HER2 in cell lines and with an epithelialphenotype indicates that differential methylation of this region couldserve as a predictive biomarker for inhibitors of EGFR or HER2signaling.

Example 4 Hypomethylation of the ERBB2 DMR Correlates with ErlotinibSensitivity

ERBB2 hypomethylation was strongly correlated with erlotinib sensitivityin vitro, indicating its potential as a predictive clinical biomarker oferlotinib response. FIG. 5 shows the results of a ERBB2 pyrosequencinganalysis of NSCLC cell lines indicating this correlation between ERBB2hypomethylation and erlotinib sensitivity. Data in this FIG. 5 isplotted as the mean+/−SD of methylation of 6 CpG sites against erlotinibsensitivity. For erlotinib IC₅₀ determination, cells were plated inquadruplicate at 3×10² cells per well in 384-well plates in RPMIcontaining 0.5% FBS (assay medium) and incubated overnight. 24 hourslater, cells were treated with assay medium containing 3 nM TGFα anderlotinib at a dose range of 10 μM-1 pM final concentration. After 72hrs, cell viability was measured using the Celltiter-Glo LuminescentCell Viability Assay (Promega). The concentration of erlotinib resultingin the 50% inhibition of cell viability was calculated from a4-parameter curve analysis and was determined from a minimum of 2experiments.

Example 5 Hypomethylation of the ERBB2 DMR Correlates to anEpithelial-Like Phenotype FFPE Tissue Samples

Fresh-frozen samples are not typically obtained during diagnosis ofNSCLCs or as part of lung cancer clinical trials. Therefore, to beamenable to clinical applications, a pyrosequencing assay must be ableto amplify limited, degraded DNA from formalin-fixed, paraffin-embedded(FFPE) tissue (commonly <150 bp). Because of the high concordancebetween the methylation states of 6 adjacent CpG sites within the ERBB2DMR using a 228-bp pyrosequencing assay (FIG. 6), the assay wasredesigned to examine just 2 CpG sites. In this assay, pyrosequencingwas used to determine quantitative methylation status of the DMR inNSCLC cell lines. Quantitative methylation was determined at 6consecutive CpG sites by Pyromark analysis software using the equation %methylation=(C peak height×100/C peak height+T peak height). FIG. 6shows the mean percent methylation of 6 individual CpG sites, with aP-value of p<0.06 determined using a Student's t-test. The designationof the cell lines as epithelial or mesenchymal was previously determinedusing a 20-gene Fluidigm Gene Expression panel.

Example 6 Hypomethylation of the ERBB2 DMR Correlates with anEpithelial-Like Phenotype in NSCLC Primary Tumors

The methylation status of ERBB2 was then evaluated in 42 late-stage(stage IIIb/IV) FFPE NSCLC tumors for which gene expression data werealso available. Hypomethylation of the ERBB2 enhancer correlatedstrongly with expression of HER2 in biopsies obtained from patients wholater went on to fail front-line chemotherapy (P<0.011), recapitulatingthe pattern observed in cell lines (FIG. 7). Hypomethylation wasdetermined using pyrosequencing and TaqMan-based Fluidigm gene expressanalysis. Percentage of methylation is represented as the mean of 2 CpGsites. A median cutoff point was used to dichotomize ERBB2-high andERBB2-low tumors. P value was determined using a one-tailed Mann-WhitneyU test.

Analysis of methylation of ERBB2 and epithelial/mesenchymal status in 47NSCLC primary tumor samples derived from archival FFPE slides wasperformed. Methylation status of ERBB2 was determined usingpyrosequencing analysis. Tumors that were classified as epithelial-likewere hypomethylated at the ERBB2 enhancer relative to tumors classifiedas mesenchymal-like (P<0.046), indicating a strong association betweenERBB2 methylation status and overall gene expression phenotype, FIG. 8(data are represented as the mean of 2 CpG cites.Epithelial-like/mesenchymal-like status was determined using scoresderived from TaqMan-based Fluidigm gene expression analysis. A mediancutoff point was used to dichotomize epithelial-like/mesenchymal-likeexpression scores. P value was determined using a Student t test).

What is claimed is:
 1. A method of determining the sensitivity of tumorcell growth to inhibition by an EGFR kinase inhibitor, comprisingdetecting the methylation status of the ERBB2 gene in a sample tumorcell, wherein hypomethylation of the ERBB2 gene indicates that the tumorcell growth is sensitive to inhibition with the EGFR inhibitor.
 2. Amethod of identifying a cancer patient who is likely to benefit fromtreatment with an EFGR inhibitor comprising detecting the methylationstatus of the ERBB2 gene from a sample from the patient's cancer,wherein the patient is identified as being likely to benefit fromtreatment with the EGFR inhibitor if the methylation status of the ERBB2gene is detected to be hypomethylation.
 3. The method of claim 1,wherein the methylation status is detected in a part of the ERBB2 gene.4. The method of claim 3, wherein the part of the ERBB2 gene is anenhancer.
 5. The method of claim 3, wherein the part of the ERBB2 geneis an enhancer and a promoter.
 6. The method of claim 3, wherein thepart of the ERBB2 gene comprises a 6 CpG repeat region.
 7. The method ofclaim 3, wherein the part of the ERBB2 gene comprises the nucleic acidsequence of SEQ ID NO:1.
 8. The method of claim 6, wherein the part ofthe ERBB2 gene comprises the nucleic acid sequence of SEQ ID NO:
 2. 9.The method of claim 1, wherein hypomethylation is indicated by less thanabout 50% methylation of the ERBB2 gene.
 10. The method of claim 9,where hypomethylation is indicated by less than about 20% methylation ofthe ERBB2 gene.
 11. The method of claim 3, wherein hypomethylation isindicated by less than about 50% methylation of the part of the ERBB2gene.
 12. The method of claim 11, wherein hypomethylation is indicatedby less than about 20% methylation of the part of the ERBB2 gene. 13.The method of claim 2, further comprising administering to the patient atherapeutically effective amount of an EGFR inhibitor if the patient isidentified as one who will likely benefit from treatment with the EGFRinhibitor.
 14. A method of treating a cancer in a patient comprisingadministering a therapeutically effective amount of an EGFR inhibitor tothe patient, wherein the patient, prior to administration of the EGFRinhibitor, was diagnosed with a cancer which exhibits hypomethylation ofthe ERBB2 gene, wherein the hypomethylation of the ERBB2 gene isindicative of therapeutic responsiveness by the subject to the EGFRinhibitor.
 15. A method of selecting a therapy for a cancer patient,comprising a. detecting the methylation status of the ERBB2 gene from asample from the patient's cancer, and b. selecting an EGFR inhibitor forthe therapy when the ERBB2 gene is detected to be hypomethylated. 16.The method of claim 15, further comprising administering to the patienta therapeutically effective amount of the EGFR inhibitor.
 17. The methodof claim 16, wherein the EGFR inhibitor is erlotinib, cetuximab, orpanitumumab.
 18. A method of determining overexpression of ERBB2 gene ina cell comprising detecting the methylation status of the ERBB2 gene inthe cell, wherein ERBB2 gene hypomethylation indicates overexpression ofERBB2 in the cell.
 19. A method of treating a cancer in a patientcomprising administering a therapeutically effective amount of a HER2inhibitor to the patient, wherein the patient, prior to administrationof the HER2 inhibitor, was diagnosed with a cancer which exhibitshypomethylation of the ERBB2 gene, wherein the hypomethylation of theERBB2 gene is indicative of therapeutic responsiveness by the subject tothe HER2 inhibitor.
 20. The method of claim 1, wherein the methylationstatus is detected by pyrosequencing.
 21. The method of claim 1, whereinthe ERBB2 gene is from a formalin-fixed paraffin embedded (FFPE) tissueor from fresh frozen tissue.
 22. The method of claim 21, wherein theERBB2 gene isolated from the tissue sample is preamplified beforepyrosequencing.